EP2938360B1 - Minimal toxische prodrugs - Google Patents

Minimal toxische prodrugs Download PDF

Info

Publication number
EP2938360B1
EP2938360B1 EP13820778.2A EP13820778A EP2938360B1 EP 2938360 B1 EP2938360 B1 EP 2938360B1 EP 13820778 A EP13820778 A EP 13820778A EP 2938360 B1 EP2938360 B1 EP 2938360B1
Authority
EP
European Patent Office
Prior art keywords
dox
prodrug
drug
seq
phac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP13820778.2A
Other languages
English (en)
French (fr)
Other versions
EP2938360A2 (de
Inventor
André Trouet
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
COBIORES NV
Original Assignee
Cobiores Nv
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cobiores Nv filed Critical Cobiores Nv
Priority to PL13820778T priority Critical patent/PL2938360T3/pl
Publication of EP2938360A2 publication Critical patent/EP2938360A2/de
Application granted granted Critical
Publication of EP2938360B1 publication Critical patent/EP2938360B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention relates to the field of oligopeptide prodrugs that are intended for the treatment of cancer.
  • the selectivity of these prodrugs requires the presence of an (oligo)peptidic moiety and/or a protective capping group to ensure the prodrug stability in blood.
  • It further in particular relates to the exemplary oligopeptidic moiety ALGP and to prodrugs comprising it.
  • chemotoxic drugs are their low selectivity for cancer cells resulting in dose limiting and life threatening toxic side effects.
  • the most common acute toxicity is myelotoxicity resulting in a severe leukopenia and thrombocytopenia.
  • Some of the commonly used drugs have also a more specific toxicity.
  • Doxorubicin (Dox) an anthracycline drug, is an 17 of such a chemotoxic drug that induces besides severe myelotoxicity a severe cardiotoxicity. These toxicities restrict its use above a cumulative dose of 500 mg/m 2 .
  • a tumor-recognizing molecule e.g. receptor ligand; see, e.g., Safavy et al. 1999 - J Med Chem 42,4919-4924
  • a peptide that is cleaved preferentially in the immediate vicinity of tumor cells by proteases preferentially secreted or produced by tumor cells e.g., a tumor-recognizing molecule, e.g. receptor ligand; see, e.g., Safavy et al. 1999 - J Med Chem 42,4919-4924 .
  • Tumor specific oligopeptidic prodrugs such as prodrugs of doxorubicin
  • these peptidic prodrugs were designed to be impermeable to cell membranes, to remain stable in the blood while being cleaved into the active drug by peptidases released in the extracellular space of solid tumors.
  • These activating peptidases are not necessarily tumor specific but can increase the drug selectivity to the extent that these peptides are oversecreted in the extracellular space of solid tumors and play an important role in cancer cell invasion and metastasis.
  • the originality of this approach is that it didn't target a single well known enzyme but all enzymatic activity that was found excreted by human tumoral cells maintained in culture.
  • Suc- ⁇ ALAL-dox The main limitation of Suc- ⁇ ALAL-dox is that leukopenia remains as an important toxicity and that experimentally higher antitumoral activity could only be observed at the cost of a still important myelotoxicity. Such myelotoxicity is expected to occur as the result from the sensitivity of the peptidic moiety of Suc- ⁇ ALAL-dox to the hydrolysis by enzymes present in normal tissues..
  • WO 02/100353 specifically discloses chemotherapeutic prodrugs designed with a 3- to 6-amino acid oligopeptide cleavable by CD10.
  • WO 02/00263 discloses prodrugs with a 3-amino acid oligopeptide cleavable by TOP and at least 1 prodrug with an amino acid oligopeptide (Leu-Ala-Gly) not cleavable by CD10.
  • WO 00/33888 and WO 01/95945 disclose prodrugs with a 4- to 20-amino acid oligopeptide comprising a non-genetically encoded amino acid at a fixed position, with said oligopeptide being cleavable by TOP.
  • WO 01/95945 at least 1 prodrug, with a ⁇ Ala-Leu-Tyr-Leu oligopeptide, was reported to be resistant to CD10 proteolytic action.
  • WO 01/95943 discloses prodrugs with a 3- to 4-amino acid oligopeptide comprising a fixed isoleucine, said oligopeptide preferably being resistant to TOP; no information on CD10-susceptibility or -resistance is given.
  • a more general concept of a prodrug consisting of a drug linked to an oligopeptide (of at least 2 amino acids) itself linked to a terminal group is disclosed in WO 96/05863 and was later extended in WO 01/91798 .
  • WO 02/07770 Other polymeric drug-conjugates of which the non-drug moiety is at least comprising a watersoluble polymer and a peptide (comprising 4 to 5 natural or non-natural amino acids) selectively cleavable by action of matrix metalloproteinases (MMPs) are disclosed in WO 02/07770 .
  • MMPs matrix metalloproteinases
  • WO 03/094972 focuses on anti-tumor prodrugs that are activatable by the human fibroblast activation protein (FAP ⁇ ); the prodrug comprises an oligopeptide of 4 to 9 amino acids with a cyclic amino acid at a fixed position.
  • WO 99/28345 discloses prodrugs that are proteolytically cleavable by prostate-specific antigen in the oligopeptide of less than 10 amino acids present in the prodrug.
  • WO 97/34927 revealed the FAP ⁇ -scissable prodrugs Ala-Pro-7-amino-4-trifluoromethylcoumarin and Lys-Pro-7-amino-4-trifluoromethylcoumarin.
  • WO 00/71571 focuses on FAP ⁇ -scissable prodrugs, with some further experimental investigations on proteolytic sensitivity to CD26 (dipeptidylpeptidase IV), the latter being considered as undesirable due to the relative abundance of CD26 also in non-malignant cells.
  • prodrugs activatable by FAP ⁇ include promellitin toxin ( LeBeau et al. 2009, Mol Cancer Ther 8, 1378-1386 ), doxorubicin ( Huang et al. 2011, J Drug Target 19, 487-496 ), thapsigargin ( Brennen et al. 2012, J Natl Cancer Inst 104, 1320-1334 ), and a prodrugs comprising an oligopeptide of 4 to 9 amino acids with a cyclic amino acid at a fixed position ( WO 03/094972 ).
  • WO 01/68145 discloses MMP-cleavable but neprilysin (CD10)-resistant doxorubicin prodrugs (see Example 1001 therein) comprising a 3- to 8-amino acid oligopeptide.
  • Metalloproteinase- and plasmin-sensitive doxorubicin prodrugs have been developed, as well as CNGRC-peptide conjugates with doxorubicin ( Hu et al. 2010, Bioorg Med Chem Lett 20, 853-856 ; Chakravarty et al. 1983, J Med Chem 26, 638-644 ; Devy et al. 2004, FASEB J 18, 565-567 ; Vanhensbergen et al. 2002, Biochem Pharmacol 63, 897-908 ).
  • WO97/12624 discloses peptide-comprising prodrugs wherein the peptide is cleavable by the prostate-specific antigen (PSA).
  • PSA prostate-specific antigen
  • prodrugs are the presence of a protecting or capping moiety, usually covalently linked to the N-terminal side of the oligopeptide, which adds to the stability of the prodrug and/or adds to the prevention of internalization of the prodrug into a cell such as a target cell.
  • protecting or capping moieties include non-natural amino acids, ⁇ -alanyl or -succinyl groups (e.g. WO 96/05863 , US 5,962,216 ).
  • Further stabilizing protecting or capping moieties include diglycolic acid, maleic acid, pyroglutamic acid, glutaric acid, (e.g., WO 00/33888 ), a carboxylic acid, adipic acid, phthalic acid, fumaric acid, naphthalene dicarboxylic acid, 1,8-naphtyldicarboxylic acid, aconitic acid, carboxycinnamic acid, triazole dicarboxylic acid, butane disulfonic acid, polyethylene glycol (PEG) or an analog thereof (e.g., WO 01/95945 ), acetic acid, 1- or 2-naphthylcarboxylic acid, gluconic acid, 4-carboxyphenyl boronic acid, polyethylene glycolic acid, nipecotic acid, and isonipecotic acid (e.g., WO 02/00263 , WO 02/100353 ), succinylated poly
  • a new type of protecting or capping moiety was introduced in WO 2008/120098 , being a 1,2,3,4 cyclobutanetetracarboxylic acid.
  • the protecting or capping moiety in WO 02/07770 may be polyglutamic acid, carboxylated dextranes, carboxylated polyethylene glycol or a polymer based on hydroxyprolyl-methacrylamide or N-(2-hydroxyprolyl)methacryloylamide.
  • the invention is defined in the claims.
  • the prodrugs of the invention have the general structure: [C x -OP] y -D, wherein
  • the oligopeptide moiety in the above structure is a tetrapeptide moiety with the sequence Ala-Leu-Gly-Pro (3-letter code), also referred to as ALGP (1-letter code; SEQ ID NO:1); or Ala-Leu-Ala-Leu (3-letter code), also referred to as ALAL (1-letter code; SEQ ID NO:2), and/or the capping group C in the above structure is a phosphonoacetyl group, and/or the drug in the above structure is doxorubicin (hereinafter also referred to as DOX or Dox).
  • ALGP Ala-Leu-Gly-Pro
  • ALAL Ala-Leu-Ala-Leu
  • the capping group C in the above structure is a phosphonoacetyl group
  • the drug in the above structure is doxorubicin (hereinafter also referred to as DOX or Dox).
  • the structure of the tetrapeptide is ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10).
  • the structure of the tetrapeptide in the above general structure is selected from the group consisting of ALGP (SEQ ID NO:1), ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), and KLKP (SEQ ID NO:10).
  • the tetrapeptide in the above general structure is selected from ALGP (SEQ ID NO:1), or KLGP (SEQ ID NO:6); even more in particular the tetrapeptide in the above general structure is ALGP (1-letter code; SEQ ID NO:1).
  • said linker or spacing group in the above prodrugs or salts thereof may be a self-eliminating linker or spacing group.
  • the invention further relates to compositions comprising one of the above prodrugs or salts thereof, or a combination of any thereof, and at least one of a solvent, diluent or carrier.
  • the invention encompasses the above prodrug or salt thereof, or the above composition comprising it for use in the treatment of a cancer.
  • Methods of treating a cancer are also part of the invention, said methods comprising administering to a subject having cancer said prodrug or salt thereof or said composition, said administering resulting in the treatment of said cancer.
  • the effective amounts of said prodrug or salt thereof, or of said composition is not causing severe leukopenia or cardiac toxicity.
  • the invention further includes methods of producing and of screening candidate prodrugs, such candidate prodrugs having the general structure [C x -OP] y -D, wherein
  • the candidates to be tested have the general structure [C x -OP] y -D, wherein
  • said screening are methods of screening candidate prodrugs having the general structure [C x -OP] y -D, wherein
  • the candidates to be tested have the general structure [C x -OP] y -D, wherein
  • screening include methods of screening candidate prodrugs having the general structure [C x -OP] y -D, wherein
  • capping group C may be a phosphonoacetyl group.
  • said drug D may be selected from the group consisting of maytansine, geldanamycin, paclitaxel, docetaxel, campthothecin, vinblastine, vincristine, vindesine, methothrexate, aminopterin, amrubicin, or a derivative of any thereof.
  • said linker or spacing group may be a self-eliminating linker or spacing group.
  • kits comprising a container comprising an above-described prodrug or salt thereof or comprising a composition comprising such prodrug or salt thereof.
  • the present invention describes new prodrug compounds of therapeutic agents, especially prodrugs comprising an antitumor therapeutic agent, displaying improved therapeutic properties.
  • the improved therapeutic properties include decreased toxicity and increased efficacy.
  • the prodrugs display a high specificity of action, a reduced toxicity, an improved stability in the serum and blood, and their therapeutic agent is not moving into target cells until the prodrug is finally activated (the activation may involve multiple steps) by (a) target cell associated enzyme(s) such as extracellularly peptidases released from the target cells or such as associated with the extracellular surface of the target cells.
  • Target cells include cancer cells as well as tumor stroma cells.
  • the prodrug compounds of the invention are prodrug forms of a therapeutic agent, in which the therapeutic agent is linked directly or indirectly to an oligopeptide, which in turn, is linked to a stabilizing capping group.
  • the prodrugs of the invention have the following general structure: [C x -OP] y -D, wherein
  • the following prodrugs are included in the general structure: C-OP-D-OP-C ; C-OP-OP-D and C-OP-D-OP.
  • the capping group C thus can be present (via direct or indirect linkage as described above) on one or more oligopeptide moieties OP in the case of a prodrug compound/molecule comprising multiple oligopeptide moieties OP.
  • the oligopeptidic moiety is a peptide with a minimum length of 4 consecutive amino acids (tetrapeptide) and a maximum length of 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino acids) which comprises a carboxy-terminal proline, wherein said proline is linked directly or via a linker or spacing group to the drug D.
  • this oligopeptidic moiety is cleaved/removed from the drug D in a two-step process wherein the first step converts said prodrug to a dipeptide-drug intermediate and wherein the second step converts the dipeptide-drug intermediate to the free drug D.
  • the dipeptide remaining in the the dipeptide-drug intermediate has the sequence glycine-proline (GP), alanine-proline (AP), or lysine-proline (KP). It is thus an object of the present invention to provide the produgs with the above-mentioned general formula, wherein OP represents a peptide with a minimum length of 4 consecutive amino acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.
  • a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino acids which comprises carboxy-terminally a proline comprising dipeptide selected from the group consisting of glycine-proline (GP), alanine-proline (AP), and lysine-proline (KP), wherein the proline of said proline comprising dipeptide is linked directly or via a linker or spacing group to the drug D.
  • GP glycine-proline
  • AP alanine-proline
  • KP lysine-proline
  • the oligopeptide moiety in the above structure is a tetrapeptide moiety with the sequence Ala-Leu-Gly-Pro (3-letter code), also referred to as ALGP (1-letter code; SEQ ID NO:1); or Ala-Leu-Ala-Leu (3-letter code), also referred to as ALAL (1-letter code; SEQ ID NO:2), and/or the capping group C in the above structure is a phosphonoacetyl group, and/or the drug in the above structure is doxorubicin (hereinafter also referred to as DOX or Dox).
  • ALGP Ala-Leu-Gly-Pro
  • ALAL Ala-Leu-Ala-Leu
  • the capping group C in the above structure is a phosphonoacetyl group
  • the drug in the above structure is doxorubicin (hereinafter also referred to as DOX or Dox).
  • the structure of the tetrapeptide is ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10).
  • the structure of the tetrapeptide in the above general structure is selected from the group consisting of ALGP (SEQ ID NO:1), ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), and KLKP (SEQ ID NO:10).
  • the tetrapeptide in the above general structure is selected from ALGP (SEQ ID NO:1), or KLGP (SEQ ID NO:6); even more in particular the tetrapeptide in the above general structure is ALGP (1-letter code; SEQ ID NO:1).
  • the drug (D) in the above general structure is doxorubicin or a pharmaceutically acceptable salt thereof.
  • the prodrug may have the structure of Compound I (see Example 3.1) or may be a pharmaceutically acceptable salt thereof.
  • the capping group phosphonoacetyl provides the advantages of avoiding the use of a non natural amino acids at the terminal end of the oligopeptide.
  • phosphonoacetyl as capping group C has the further advantage of providing a negatively charge that is important in order to avoid aggregation of oligopeptide derivatives of doxorubicin.
  • the stability of a prodrug of the invention can be defined such that less than 10% of cleavage derivatives are to be obtained upon incubation of the prodrug in human blood for more than 2 hours.
  • the oligopeptide moiety OP and the drug are not limiting the current invention and other combinations are envisaged by the invention. These combinations include any OP and/or D with the capping group C being a phosphonoacetyl group. Further combinations include any C (any capping group known in the art) and/or any D with the oligopeptide moiety OP being a tetrapeptide moiety with the sequence Ala-Leu-Gly-Pro (3-letter code) or Ala-Leu-Ala-Leu (3-letter code) or ALGP (1-letter code; SEQ ID NO:1) or ALAL (1-letter code; SEQ ID NO:2).
  • the structure of the tetrapeptide is ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10).
  • the structure of the tetrapeptide is ALGP (SEQ ID NO:1), ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10).
  • the tetrapeptide in the above general structure is selected from ALGP (SEQ ID NO:1), or KLGP (SEQ ID NO:6); even more in particular the tetrapeptide in the above general structure is ALGP (1-letter code; SEQ ID NO:1).
  • oligopeptidic moieties of the invention can be linked to the drug.
  • the oligopeptidic moieties can themselves form a linear or branched structure linked to the drug or, alternatively, multiple oligopeptidic moieties are each individually linked to the drug.
  • the capping group or protecting or capping moiety is linked to the oligopeptidic moiety of the prodrug and adds to the solubility and/or stability of the prodrug (e.g. in blood of the animal, mammal, human or subject to which the prodrug is administered) and/or adds to the prevention of internalization of the prodrug into a cell such as a target cell.
  • the linkage between the capping group and the oligopeptide and/or between the oligopeptide and the therapeutic agent or drug may be direct, e.g. via the N-terminal aminogroup of the oligopeptide or the C-terminal carboxylgroup of the oligopeptide, or via a side chain of one of the amino acids of the oligopeptide.
  • said linkage may be indirect, e.g. by introducing a linker or spacer group between the oligopeptide OP and the drug D.
  • a linker between D and OP is not required per se as enzymatic scission of the amide bond between D and OP ensures the availability of the free NH 2 -group on the cytoxic drug.
  • cytotoxic drugs D do not have any free NH 2 -group and cannot as such be linked to OP by an amide bond. Introducing an NH 2 -group to those molecules may decrease or suppress their cytotoxic activity.
  • a self-immolating (or self-eliminating, self-sacrificing, self-lysing, or self-leaving) spacing group or spacer can be used as linker between the drug D and the oligopeptide OP.
  • OP is linked to the self-immolating spacer by an amide bond sensitive to extracellular enzymes capable of activating the prodrug.
  • Self-immolating spacer After cleavage of the amide bond between OP and the spacer, the self-immolating spacer cleaves itself from the drug leaving it underivatized, i.e., leaving it in its original active form.
  • Self-immolative spacers include para-aminobenzoyloxycarbonyl moieties that are able to connect either -OH, - COOH, - NH, or -SH groups of a drug at the one hand to the carboxy-terminal group of a peptide at the other hand.
  • This type of linker is an electronic cascade spacer. Such bond has been shown to be cleavable by peptidases.
  • linker or spacer groups examples include aminocaproic acid, a hydrazide group, en ester group, an ether group and a sulphydryl group.
  • a linker or spacer group as described above between the capping group and the oligopeptide and/or between the oligopeptide and the therapeutic agent may be advantageous for reasons such as the following: (i) as a spacer for steric considerations in order to facilitate enzymatic release of the amino acid linked to the therapeutic agent or other enzymatic activation steps; (ii) to provide an appropriate attachment chemistry between the different moieties of the prodrug (and thus providing flexibility to couple any possible drug and/or capping moiety to the oligopeptide of the invention); (iii) to improve the synthetic process of making the prodrug conjugate (e.g., by pre-derivatizing the therapeutic agent or oligopeptide with the linker group before conjugation to enhance yield or specificity); (iv) to improve
  • the linkage should: (1) not or not significantly disturb the functionality of the oligopeptidic moiety, i.e., should not significantly disturb neither the proteolytic scissability by TOP nor the resistance to proteolytic scissability by CD1 and (2) retain the blood stability of the compound. Determination of the functionality of the capped oligopeptidic moiety in the prodrug can be tested easily and in a straightforward way, e.g. as described in the Examples section hereafter. Such testing does not amount to an undue burden for a skilled person.
  • pharmaceutically acceptable salt(s) means those salts of compounds of the invention that are safe and effective for the intended medical use that possess the desired biological activity.
  • Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • bases salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • the drug or therapeutic agent conjugated to the oligopeptide of the invention may be useful for treatment of cancer (e.g. by exerting cytotoxic or antiangiogenic activity), inflammatory disease, or some other medical condition.
  • the drug or therapeutic agent conjugated to the oligopeptide of the invention may be any drug or therapeutic agent capable of entering a target cell.
  • the therapeutic agent may be selected from a number of classes of compounds including, antibiotics, alkylating agents, antiproliferative agents, tubulin binding agents, vinca alkaloids, enediynes, podophyllotoxins or podophyllotoxin derivatives, the pteridine family of drugs, taxanes, anthracyclines, dolastatins, topoisomerase inhibitors, platinum-coordination-complex chemotherapeutic agents, and maytansinoids.
  • said drug or therapeutic agent may be one of the following compounds, or a derivative or analog thereof: doxorubicin, daunorubicin, amrubicin, vinblastine, vincristine, calicheamicin, etoposide, etoposide phosphate, CC-1065, duocarmycin, KW 20 2189, methotrexate, methopterin, aminopterin, dichloromethotrexate, docetaxel, paclitaxel, epithiolone, combretastatin, combretastatin A4 phosphate, dolastatin 10.
  • dolastatin 11, dolastatin 15, topotecan camptothecin, mitomycin C, porfiromycin, 5 fluorouracil, 6-mercaptopurine, fludarabine, tamoxifen, cytosine arabinoside, adenosine arabinoside, colchicine, halichondrin B, cisplatin, carboplatin, mitomycin C, bleomycin, melphalan, chloroquine, cyclosporin A, and maytansine.
  • derivative is intended a compound that results from reacting the named compound with another chemical moiety (different from the oligopeptidic moiety linked directly or indirectly to the compound), and includes a pharmaceutically acceptable salt, acid, base or ester of the named compound.
  • Other therapeutic agents or drugs include: vindesine, vinorelbine, 10-deacetyltaxol, 7-epi-taxol, baccatin III, 7-xylosyltaxol, isotaxel, ifosfamide, chloroaminophene, procarbazine, chlorambucil, thiophosphoramide, busulfan, dacarbazine (DTlC), geldanamycin, nitroso ureas, estramustine, BCNU, CCNU, fotemustine, streptonigrin, oxaliplatin, methotrexate, aminopterin, raltitrexed, gemcitabine, cladribine, clofarabine, pentostatin, hydroxyureas, irinotecan, topotecan, 9-dimethylaminomethyl-hydroxy-camptothecin hydrochloride, teniposide, amsacrine; mitoxantrone;
  • Example 16 For a number of compounds included in the above listing, more experimental guidance is given in Example 16 herein.
  • amrubicin which is an anthracycline analogue with a free NH 2 -group ( Hanada et al. 1998, Jpn J Cancer Res 89, 1229-1238 ) that can be linked to a capped oligopeptide such as PhAc-ALGP by the same method as used for doxorubicin.
  • Addition of (a) polyethylene glycol group(s) to the amino- of the oligopeptidic moiety) may be performed in order to increase the half-life of the prodrug according to the invention in circulation after administration to a mammal. Addition of (a) polyethylene glycol group(s) could also play the role of a capping agent.
  • a prodrug or salt thereof of the invention can further be present in a composition
  • a composition comprising besides the prodrug or salt thereof any one of a suitable solvent (capable of solubilizing the prodrug to the desired extent), diluent (capable of diluting concentrated prodrug to the desired extent) or carrier (any compound capable of absorbing, adhering or incorporating the prodrug, and of subsequently releasing at any rate the prodrug in the extracellular compartment of the subject's body).
  • Said composition may alternatively comprise multiple (i.e. more than 1) prodrug or salt thereof, or any combination thereof (e.g.
  • said solvent, diluents or carrier is pharmaceutically acceptable, i.e., is acceptable to be administered to a subject to be treated with the composition of the invention. Aiding in formulating a pharmaceutically acceptable composition is e.g. any Pharmacopeia book.
  • the composition may be formulated such that it is suitable for any way of administration including intra-cranial, intra-spinal, enteral and parenteral administration.
  • the regimen by which the prodrug is administered may vary, e.g. depending on whether or not a capping group is present, depending on the formulation, depending on the overall physical condition of a subject to be treated and e.g. depending on the judgment of the treating physician.
  • the prodrug or salt thereof of the invention, or a composition comprising it, is particularly suitable for treating a disease that is treatable by the released drug.
  • cancer or tumors such as solid tumors.
  • "Cancer” includes e.g. breast cancers, colorectal cancers, liver cancers, lung cancers such as small cell, non-small cell, bronchic cancers, prostate cancers, ovarian cancers, brain cancers, and pancreatic cancers, colon cancers, head and neck cancers, stomach cancers, bladder cancers, non-Hodgkin's lymphomas, melanomas, leukaemias, neuroblastomas, and glioblastomas.
  • the subject to be treated with the prodrug of the invention can be any mammal in need of such treatment but is in particular a human.
  • the treatment can result in regression of the disease (e.g. in terms of decreasing tumor volume or tumor mass and of metastases), in decreased progression of the disease compared to expected disease progression, or in stabilization of the disease, i.e. neither regression nor progression of the disease. All these are favorable outcomes of the treatment.
  • the effective amounts of said prodrug or salt thereof, or of said composition is not causing severe leukopenia or cardiac toxicity/cardiotoxicity.
  • a possible definition of severe human leukopenia is WHO-criteria-defined grade 3- (1000-1900 leukocytes/mL) or grade 4-leukopenia (less than 1000 leukocytes/mL).
  • an anticancer prodrug (or a salt thereof) according to the present invention in combination therapies is also envisaged.
  • This can be in a combined modality chemotherapy, i.e. the use of the anticancer prodrug (or a salt thereof) with other cancer treatments, such as radiation therapy (whether by direct irradiation or via administering an isotope-labeled antibody or antibody fragment) or surgery.
  • This can also be in combination chemotherapy, i.e. treating a patient with a number of different drugs wherein the drugs preferably differ in their mechanism of action and in their side effects.
  • the drugs are administered simultaneously.
  • An advantage of combination chemotherapy is the minimization of the chance of the development of resistance to any one agent.
  • a further advantage may be that the individual drugs can each be used at a lower dose, thereby reducing overall toxicity.
  • a prodrug or salt thereof according to the invention, or a composition comprising such prodrug or salt can thus be used for treatment of a disease (e.g. cancer), as monotherapy, or as part of a combination chemotherapy treatment or a combined modality chemotherapy treatment.
  • an anticancer prodrug (or a salt thereof) according to the invention can be combined with one or more alkylating antineoplastic agent(s) and/or one or more anti-metabolite(s) and/or one or more anti-microtubule agent(s) and/or one or more topoisomerase inhibitor(s) and/or one or more cytotoxic antibiotic(s) and/or one or more biological anticancer agent(s) (such as antibodies), wherein one or more of these, when applicable, can also be prodrug(s) (or a salt thereof) according to the present invention.
  • the drug doxorubicin (also known under the trade names Adriamycin or Rubex) is commonly used to treat multiple types of cancers such as some leukemias and Hodgkin's lymphoma, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma, and others. Doxorubicin is further used in different combination therapies.
  • Doxorubicin-containing therapies include AC or CA (Adriamycin, cyclophosphamide), TAC (Taxotere, AC), ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin (doxorubicin), cyclophosphamide, Oncovin (vincristine), procarbazine, prednisone), CHOP (cyclophosphamide, Adriamycin, vincristine, prednisolone), FAC or CAF (5-fluorouracil, Adriamycin, cyclophosphamide), MVAC (methothrexate, vincristine, adriamycin, cisplatin), CAV (cyclophosphamide, doxorubicin, vincristine) and CAVE (CAV, etoposide), CVAD (cyclophosphamide,
  • combination chemotherapies such as BEP (Bleomycin, etoposide, platinum agent (cisplatin (Platinol))), CAPOX or XELOX (capecitabine, oxaliplatin), CBV (cyclophosphamide, carmustine, etoposide), FOLFIRI (fluorouracil, leucovorin, irinotecan), FOLFIRINOX (fluorouracil, leucovorin, irinotecan, oxaliplatin), FOLFOX (fluorouracil, leucovorin, oxaliplatin), EC (epirubicin, cyclophosphamide), ICE (ifosfamide, carboplatin, etoposide (VP-16)) and IFL (irinotecan, leucovorin, fluorouracil).
  • BEP Boleomycin, etoposide, platinum agent (cisplatin (Platinol)
  • combination therapies including an anticancer prodrug (or a salt thereof) according to the invention (whether alone or already part of a combination chemotherapy or of a combined modality therapy) and compounds other than cytostatics.
  • Such other compounds include any compound approved for treating cancer or being developed for treating cancer.
  • such other compounds include monoclonal antibodies such as alemtuzumab (chronic lymphocytic leukemia), bevacizumab (colorectal cancer), cetuximab (colorectal cancer, head and neck cancer), denosumab (solid tumor's bony metastases), gemtuzumab (acute myelogenous leukemia), ipilimumab (melanoma), ofatumumab (chronic lymphocytic leukemia), panitumumab (colorectal cancer), rituximab (Non-Hodgkin lymphoma), tositumomab (Non-Hodgkin lymphoma) and trastuzumab (breast cancer).
  • monoclonal antibodies such as alemtuzumab (chronic lymphocytic leukemia), bevacizumab (colorectal cancer), cetuximab (colorectal cancer, head and neck cancer), denosumab (solid
  • antibodies include for instance abagovomab (ovarian cancer), adecatumumab (prostate and breast cancer), afutuzumab (lymphoma), amatuximab, apolizumab (hematological cancers), blinatumomab, cixutumumab (solid tumors), dacetuzumab (hematologic cancers), elotuzumab (multiple myeloma), farletuzumab (ovarian cancer), intetumumab (solid tumors), Matuzumab (colorectal, lung and stomach cancer), onartuzumab, parsatuzumab, pritumumab (brain cancer), tremelimumab, ublituximab, veltuzumab (non-Hodgkin's lymphoma), votumumab (colorectal tumors), zatuximab and anti-placental growth factor antibodies such as described
  • combination therapies include for instance CHOP-R (CHOP (see above)+ rituximab), ICE-R (ICE (see above) + rituximab), R-FCM (rituximab, fludarabine, cyclophosphamide, mitoxantrone) and TCH (Paclitaxel (Taxol), carboplatin, trastuzumab).
  • alkylating antineoplastic agents include nitrogen mustards (for example mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan), nitrosoureas (for example N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine and streptozotocin), tetrazines (for example dacarbazine, mitozolomide and temozolomide), aziridines (for example thiotepa, mytomycin and diaziquone (AZQ)), cisplatins and derivatives (for example cisplatin, carboplatin and oxaliplatin), and non-classical alkylating agents (for example procarbazine and hexamethylmelamine)
  • nitrogen mustards for example mechlorethamine, cyclophosphamide, melphalan
  • Subtypes of the anti-metabolites include the anti-folates (for example methotrexate and pemetrexed), fluoropyrimidines (for example fluorouracil , capecitabine and tegafur/uracil), deoxynucleoside analogues (for example cytarabine, gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine, clofarabine and pentostatin) and thiopurines (for example thioguanine and mercaptopurine).
  • fluoropyrimidines for example fluorouracil , capecitabine and tegafur/uracil
  • deoxynucleoside analogues for example cytarabine, gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine, clofarabine and pentostatin
  • thiopurines for example thioguanine
  • Anti-microtubule agents include the vinca alkaloid subtypes (for example vincristine, vinblastine, vinorelbine, vindesine and vinflunine) and taxane subtypes (for example paclitaxel and docetaxel).
  • Other anti-microtubule agents include podophyllotoxin.
  • Topoisomerase inhibitors include topoisomerase I inhibitors (for example irinotecan, topotecan and camptothecin) and topoisomerase II inhibitors (for example etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin).
  • topoisomerase I inhibitors for example irinotecan, topotecan and camptothecin
  • topoisomerase II inhibitors for example etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.
  • Cytotoxic antibiotics include anthracyclines (doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone) and other drugs including actinomycin, bleomycin, plicamycin and mitomycin.
  • anthracyclines doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone
  • other drugs including actinomycin, bleomycin, plicamycin and mitomycin.
  • Any anticancer prodrug (or a salt thereof) according to the invention can (whether alone or already part of a combination chemotherapy or of a combined modality therapy) further be included in an antibody-directed enzyme prodrug therapy (ADEPT), which includes the application of cancer-associated monoclonal antibodies, which are linked, to a drug-activating enzyme. Subsequent systemic administration of a non-toxic agent results in its conversion to a toxic drug, and resulting in a cytotoxic effect which can be targeted at malignant cells ( Bagshawe et al. (1995) Tumor Targeting 1, 17-29 .)
  • ADPT antibody-directed enzyme prodrug therapy
  • any anticancer prodrug (or a salt thereof) according to the invention can (whether alone or already part of a combination chemotherapy or of a combined modality therapy) be combined with one or more agent(s) capable of reversing (multi)drug resistance ((M)DR reverser(s) or (M)DR reversing agent(s)) that can occur during chemotherapy.
  • agents include for example loperamide ( Zhou et al. 2011, Cancer Invest 30, 119-125 ).
  • Another such combination includes loading the prodrug in nanoparticles such as iron oxide nanoparticles ( Kievit et al. 2011, J Control Release 152, 76-83 ) or liposomes.
  • doxorubicin HCL liposomes also known under the trade names Doxil, Caelyx or Myocet
  • daunorubicin known under the trade name DaunoXome
  • paclitaxel Garcion et al. 2006, Mol Cancer Ther 5, 1710-1722 ).
  • a prodrug or salt thereof according to the invention, or a composition comprising such prodrug or salt can thus be used for treatment of a disease (e.g. cancer), as monotherapy, or as part of a combination chemotherapy treatment or a combined modality chemotherapy treatment. Any of such treatments can further be combined with a treatment including a drug resistance reverting agent.
  • the invention further relates to methods of producing the prodrugs of the invention, said methods comprising the steps of:
  • said linking of the oligopeptidic moiety with the drug and/or capping group may be direct, or indirect via a linker or spacing group, such as a self-immolating or self-eliminating spacer.
  • the purification strategy of the prodrug will obviously depend on the nature of the drug and/or of the capping group. A skilled person will be able to design a suitable purification strategy for any possible prodrug according to the invention, chosing from a plethora of purification techniques that are available.
  • the picture emerging from the Examples as described herein is one that, for the exemplary prodrug of the invention comprising the ALGP-peptide (SEQ ID NO:1) as OP moiety and doxorubicin as the drug D, the activation of the prodrug is occurring in multiple steps.
  • the prodrug is stable in blood and plasma, it is converted in a mixture of doxorubicin (Dox), GP-Dox and LGP-Dox when incubated in the presence of LS-174T tumor cells.
  • the latter process can in a first step be mimicked in vitro by proteases such as CD10 (yielding LGP-Dox that can be converted to GP-Dox by common leucine aminopeptidases) and TOP (yielding GP-Dox).
  • the first phase of the activation of the ALGP-doxorubicin is driven by the preferential activity of CD 10 and TOP in the vicinity of the tumors compared to their lower abundancy in non-pathological extracellular compartments and tissues.
  • the second step, conversion of GP-Dox to Dox can be driven by dipeptidyl prolyl peptidases. Two members of this class are of interest in the area of cancer: DPIV, also known as CD26 and FAP or fibroblast activation protein.
  • proteases are known to be associated with tumor cells or tumor stromal cells as described hereafter.
  • Such multistep activation of a prodrug of the invention increases the specificity and decreases the unwanted side effects (such as leucopenia and cardiac toxicity) compared to similar prodrugs that are activatable in a single step.
  • An example of the latter is succinyl- ⁇ ALAL-doxorubicin which is easily converted by e.g. CD10 to L-Dox that can enter the cell on its own ( Pan et al. 2003, Cancer Res 63, 5526-5531 ).
  • PhAc-ALGP-doxorubicin prodrug that is about 20 to 40 times less toxic than doxorubicin varying with the mode (IV or IP) of administration, and between 6 and 14 times less toxic than succinyl- ⁇ -ALAL-doxorubicin.
  • PhAc-ALGP-doxorubicin is devoid of chronic cumulative cardiotoxicity and does not induce leucopenia and lymphopenia. It is more active than doxorubicin on human tumor xenografts (including a sarcoma) and on an orthotopic colon carcinoma.
  • the prodrugs of the invention therefore are further characterized by being activatable, in vitro or in vivo, in at least two steps, i.e., in a process involving at least two essential proteolytic cleavages by at least two different proteases.
  • An "essential proteolytic cleavage” is herein meant to be a cleavage that is associated with a tumor or a tumor-associated cell such as its stroma, i.e., is specifically occurring in the direct vicinity of a tumor or tumor-associated cells.
  • CD10 is a neutral endopeptidase/ a zinc dependent cell surface metallopeptidase that cleaves small peptides on the amino-side of hydrophobic amino acids.
  • normal cells such as B cells and epithelial cells of the lung, colon and kidney, it is present in many tumor types ( Ravel et al. 2008, Clin Cancer Res 14, 1258-1265 ), such as pancreatic cancer ( Notohara et al. 2000, Am J Surg Pathol 24, 1361-1371 ), hepatocellular carcinoma ( Karabork et al. 2010, Pathol Res Pract 206, 572-577 ), melanoma ( Velazquez et al.
  • CD10 is expressed in nearly half of the soft tissue sarcomas (histiocytomas, fibrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, malignant peripheral nerve sheat tumors; Deniz et al.
  • CD10 is found in the stromal cells of colorectal carcinomas ( Hirano et al. 2012, Pathol Int 62,600-611 ), breast cancer ( Desmedt et al. 2012, Clin Cancer Res 18, 1004-1014 ; Marketsov et al. 20, 84-89), pancreatic endocrine tumors ( Deschamps et al. 2006, Hum Path 37, 802-808 ), gastric carcinoma ( Huang et al. 2005, Jpn J Clin Oncol 35, 245-250 ) and basal cell carcinoma ( Yada et al. 2004, Am J Dermatopathol 26, 463-473 ).
  • TOP Thimet Oligo Peptidase
  • TOP is a thiol-dependent main cytoplasmic metallo-endoprotease distributed throughout many tissues and cell types. It can attain an extracellular location both via secretion of the soluble enzyme and by attachment to the plasma membrane. It is distributed throughout many tissues and cell types. TOP is involved in neuroendocrine signaling and in the extracellular metabolism of neuropeptides ( Corie et al. 2002, Endocrine Rev 23, 647-664 ). It is involved in the metabolism of proteasomes ( Saric et al. 2004, J Biol Chem 45, 46723-46732 ). TOP is involved in neuropeptide processing in prostate and prostate cancer ( Swanson et al.
  • DPIV is a dipeptidylprolylpeptidase with a broad spectrum of activity and covers a large number of physiological substrates. It is expressed in epithelial cells of a large number of organs. It is expressed in thymus spleen and lymph nodes as well as lymphocytes. DPIV binds to collagen and fibronectin in experimental conditions ( Loster et al. 1995, Biochem Biophys Res Commun 217, 341 ). It is upregulated in the tumoral T-cell malignancies ( Dang et al. 2002, Histol Histopathol 17, 1213-1226 ) and in different adenocarcinomas, such as in hepatocellular carcinoma ( Stecca et al.
  • DPIV is expressed in cancer stem cells of human colorectal cancer and of human mesotheliomas ( Pang et al. 2010, Cell Stem Cell 6, 603-615 ; Yamazaki et al. 2012, Biochem Biophys Res Commun 419, 529-536 ).
  • FAP is a dipeptidyl exopeptidase with very narrow specificity restricted to glycine-proline, alanine-proline and lysine-proline dipeptides and is also a type I collagenase. It can however also act as endopeptidase ( Siew lai et al. 2007, Bioconj Chem 18, 1245-1250 ). FAP is absent in normal adult tissues such as epithelial, mesenchymal; neural and lymphoid cells such as lymphocytes. It is absent in non-malignant tumors.
  • the term "corresponding to” is to be understood such that the selected candidate prodrug [C x -OP] y -D is comprising the same drug D as present in the dipeptide-drug intermediate prodrug GP-D, AP-D or KP-D identified to have comparable cytotoxic activity as drug D.
  • the drug D is connected to the oligopeptide moiety OP in the same was as it is connected to the GP-, AP- or KP-dipeptide in the dipeptide-drug intermediate prodrug GP-D, AP-D or KP-D.
  • the success of the in step (v) identified dipeptide-drug intermediate prodrug GP-D, AP-D or KP-D is indicative or predictive for the success of the candidate prodrug [C x -OP] y -D
  • OP represents a peptide with a minimum length of 4 consecutive amino acids (tetrapeptide) and a maximum length of 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino acids) which comprises carboxy-terminally a proline comprising dipeptide selected from the group consisting of glycine-proline (GP), alanine-proline (AP), and lysine-proline (KP).
  • doxorubicin as drug D.
  • a dipeptide-drug intermediate prodrug GP-D, AP-D or KP-D has a cytotoxic activity comparable to the cytotoxic acitivity of drug D, this is a good indication of the successful activation of such prodrug to the free drug D by the cultured cells.
  • the cultured cells used in this type of screening may for instance be a cultured tumor cell line.
  • the peptide OP in the above mentioned general structure is a tetrapeptide with the sequence ALGP (SEQ ID NO:1), ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10).
  • ALGP SEQ ID NO:1
  • ALAP SEQ ID NO:3
  • TSGP SEQ ID NO:4
  • TSAP SEQ ID NO:5
  • KLGP SEQ ID NO:6
  • KLAP SEQ ID NO:7
  • ALKP SEQ ID NO:8
  • TSKP SEQ ID NO:9
  • KLKP SEQ ID NO:10
  • said methods are methods of screening candidate prodrugs having the general structure [C x -OP] y -D, wherein
  • the term "comparable cytotoxic activity” is to be understood such that a prodrug, after contact with the in vitro cultured cells (such as cultured tumor cells), exerts at least 50% or at least between 50 and 99 % (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 55%, at least 90%, at least 95%, at least 99%) of the cytotoxic activity exerted by the free drug contacted with the same in vitro cultured cells under the same conditions.
  • the in vitro cultured cells such as cultured tumor cells
  • said methods are methods of screening candidate prodrugs having the general structure [C x -OP] y -D, wherein
  • said methods are methods of screening candidate prodrugs having the general structure [C x -OP] y -D, wherein
  • the term "corresponding to” is to be understood such that the selected candidate prodrug [C x -OP] y -D is comprising the same drug D as present in the prodrug GP-D identified to be converted by at least 50% into D under the defined conditions.
  • the drug D is connected to the oligopeptide moiety OP in the same was as it is connected to the GP-dipeptide in the prodrug GP-D.
  • the success of the in step (v) identified prodrug GP-D is indicative or predictive for the success of the candidate prodrug [C x -OP] y -D wherein OP is the ALGP-peptide.
  • the conversion percentage used for selecting the candidate prodrug in general lies within at least 50 to 100% (e.g. at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 55%, at least 90%, at least 95%, at least 99%).
  • capping group C may be a phosphonoacetyl group.
  • the OP in any one of the above screening methods is a peptide with a minimum length of 4 consecutive amino acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.
  • a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino acids which comprises carboxy-terminally a proline comprising dipeptide selected from the group consisting of glycine-proline (GP), alanine-proline (AP), and lysine-proline (KP); more in particular a tetrapeptide with the sequence ALGP (SEQ ID NO:1), ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10); even more in particular OP is the tetrapeptide ALGP (SEQ ID NO:1).
  • said drug D may be selected from the group consisting of doxorubicin, maytansine, geldanamycin, paclitaxel, docetaxel, campthothecin, vinblastine, vincristine, vindesine, methothrexate, aminopterin, amrubicin, or a derivative of any thereof.
  • kits comprising a container comprising a prodrug or salt thereof according to the invention or comprising a composition comprising such prodrug or salt thereof.
  • kit may further comprise, in the same container (holding a prodrug or salt thereof according to the invention) or in one or more separate containers, one or more further anticancer drugs, such as an antibody or fragment thereof (e.g. as described above).
  • kit may further comprise, in the same container (holding a prodrug or salt thereof according to the invention) or in one or more separate containers, one or more drug resistance reversing agents.
  • kit include one or more diagnostic agents capable of determining the success of a therapy comprising a prodrug or salt thereof according to the invention; use instructions; one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents; one or more containers with agents for ADEPT therapy; etc.
  • the Fmoc-Leu-Wang resin (5g, 1 eq) was swollen in dimethylformamide (20 mL) for 30 minutes.
  • the Fluorenylmethyloxycarbonyl (Fmoc) -group was removed by treatment with piperidine (4 mL) in dimethylformamide (16 mL) for 3 minutes, the resin was then filtered, followed by the same treatment for 3 and 7 minutes.
  • the resin was washed with dimethylformamide (20 mL) three times.
  • Fmoc-Ala-OH (2 eq) and 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (2 eq) were solubilised in dimethylformamide (20 mL) and N,N-diisopropylethylamine (4 eq) was added. The mixture was preactivated during 6 minutes and added to the resin. The resin was then shaken for 60 minutes and washed three times with dimethylformamide (20 mL). The Fmoc group was removed by treatment with piperidine (4 mL) in dimethylformamide (16 mL) for 3 minutes, the resin was then filtered.
  • Doxorubicin (1 eq) was solubilised in dimethylformamide (10 mL).
  • a solution of Fmoc-ALAL-OH (1.2 eq) in dimethylformamide (2mL) was added to the doxorubicin and the pH was adjusted to pH 8 with N,N-diisopropylethylamine.
  • the solution was stirred at RT and the 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (1.2 eq) in dimethylformamide (2 mL) was added.
  • the pH of the solution was checked and readjusted to pH 8-8.5.
  • the solution was stirred at room temperature and was checked by HPLC.
  • the Fmoc group was removed by treatment with piperidine (10% final volume) during 5 minutes at RT and the lactate buffer 10% pH 3 was added at 0°C. The mixture was loaded on YMC. The product was recovered with MeOH and the solvent was evaporated.
  • the ALAL-Doxorubicin was purified by HPLC semi-preparative (column Luna, C18). MS (ES + ): 912 [MH] + ; Purity: 95 % (determined by HPLC at 214 nm).
  • Doxorubicin (1 eq) was solubilised in dimethylformamide (10 mL).
  • a solution of Fmoc-Proline-OH (1.2 eq) in dimethylformamide (2 mL) was added to the doxorubicin and the pH was adjusted to pH 8-8.5 with N , N -diisopropylethylamine.
  • the solution was stirred at RT and the 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (1.2 eq) in dimethylformamide (2 mL) was added.
  • the pH of the solution was checked and readjusted to pH 8-8.5.
  • Pro-Doxorubicin (1 eq) was solubilised in dimethylformamide (10 mL).
  • a solution of Fmoc-ALG-OH (1.2 eq) in dimethylformamide (2mL) was added to the doxorubicin and the pH was adjusted to pH 8 with N,N-diisopropylethylamine.
  • the solution was stirred at RT and the 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (1.2 eq) in dimethylformamide (2 mL) was added.
  • the pH of the solution was checked and readjusted to pH 8-8.5.
  • NH 2 -ALGP-Dox (1 eq) was solubilised in dimethylformamide (10 mL).
  • a solution of Phosphonoacetic acid (2.5 eq) in dimethylformamide (2mL) was added to the peptide-doxorubicin and the pH was adjusted to pH 8 with N,N-diisopropylethylamine.
  • the solution was stirred at RT and the 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (2 eq) in dimethylformamide (2 mL) was added.
  • the pH of the solution was checked and readjusted to pH 8-8.5.
  • NH 2 -ALAL-Dox (1 eq) was solubilised in dimethylformamide (10 mL).
  • a solution of Phosphonoacetic acid (2.5 eq) in dimethylformamide (2mL) was added to the peptide-doxorubicin and the pH was adjusted to pH 8 with N,N-diisopropylethylamine.
  • the solution was stirred at RT and the 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (2 eq) in dimethylformamide (2 mL) was added.
  • the pH of the solution was checked and readjusted to pH 8-8.5.
  • PhAc-D(Dmab)LGP-doxorubicin was prepared as described in 3.2 with NH2- D(Dmab)LGP - Dox instead of NH2-ALAL-Dox.
  • the protecting group Dmab also known as 4- ⁇ N-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino ⁇ benzyl, was removed with hydrazine hydrate 2% during 5 minutes at room temperature.
  • the lactate buffer 10% pH 3 was added at 0°C and the mixture was loaded on YMC. The product was recovered with MeOH and the solvent was evaporated.
  • PhAc-DLGP-Doxorubicin was purified by HPLC semi-preparative (column Luna, C18). MS (ES + ): 1048.3 [MH]+; Purity: 99 % (determined by HPLC at 214 nm).
  • PhAc-T(Dmab)SGP-doxorubicin was prepared as described in 3.2 with NH 2 -T(Dmab)SGP-Dox instead of NH 2 -ALAL-Dox.
  • the protecting group Dmab was removed with hydrazine hydrate 2% during 5 minutes at room temperature.
  • the lactate buffer 10% pH 3 was added at 0°C and the mixture was loaded on YMC.
  • the product was recovered with MeOH and the solvent was evaporated.
  • the PhAc-TSGP-Doxorubicin was purified by HPLC semi-preparative (column Luna, C18). MS (ES): 1008.3 [MH]+; Purity: 99 % (determined by HPLC at 214 nm).
  • PhAc-K(IvDde)LGP-doxorubicin was prepared as described in 3.2 with NH2- K(IvDde)LGP -Dox instead of NH 2 -ALAL-Dox.
  • the protecting group IvDde was removed with hydrazine hydrate 2% during 5 minutes at room temperature.
  • the lactate buffer 10% pH 3 was added at 0°C and the mixture was loaded on YMC.
  • the product was recovered with MeOH and the solvent was evaporated.
  • the PhAc-KLGP-Doxorubicin was purified by HPLC semi-preparative (column Luna, C18). MS (ES + ): 1061 [MH]+; Purity: 91.5 % (determined by HPLC at 214 nm).
  • Sub-confluent cultures of LS-174T tumor cells were washed twice with a saline phosphate buffer solution, and fresh culture medium (DMEM-F12 without phenol red) containing 0.02% bovine serum albumin is added (100 ⁇ l/cm 2 ). After 24 hours incubation, the conditioned medium is collected, centrifuged for 10 minutes at 300 g, buffered with 1 M Tris-HCl, pH 7.4 (1 volume of buffer + 19 volumes of medium) and concentrated 20 times by ultracentrifugation (cutoff threshold of 10 kDa). Drug compounds (50 ⁇ M) were incubated for 0, 1, 3 or 5 hours at 37°C in the presence of freshly prepared LS174T tumor cell conditioned medium. Fifty ⁇ L of sample were collected at each time point and processed as described above for human plasma.
  • CD10 (recombinant human neprilysin, R&D systems, Ref. 1182-ZN) was diluted at 20 nM in 0.1M MES pH 6.5 supplemented with 0.2 mg/mL BSA.
  • TOP (recombinant human thimet oligopeptidase, R&D systems, Ref. 3439-ZN) was diluted at 10 nM in a solution of 50 mM Tris-HCl pH 7.4/ 0.5M NaCl/ 0.1M DTT.
  • CD26 and FAP were diluted at 1 ⁇ g/mL in a Tris-HCl pH 7.5 buffer.
  • Reactions were initiated by addition of 50 ⁇ M of each compound to enzymes solutions (1 V enzyme solution + 1 V 100 ⁇ M drug solution). Samples were incubated for 0, 1, or 3 hours at 37°C in a water bath in the presence of purified enzymes. Fifty ⁇ L of sample were collected at each time point and processed as described above for human plasma. Activation of Suc- ⁇ ALAL-dox was tested in parallel as a reference.
  • Suc- ⁇ ALAL-Dox is cleaved by CD10 to release 73% of L-Dox.
  • PhAc-ALAL-Dox is not efficiently cleaved by CD10. Less than 5% of L-Dox is released after 3 hours of incubation in the presence of the enzyme.
  • replacement of the succinyl- A- by the phosphonoacetyl capping group inhibits the peptide-enzyme interaction.
  • PhAc-ALAF-Dox is moderately cleaved by CD10 into F-Dox. Changing the ALAF peptide moiety into ALPF, inhibits cleavage by CD10 whatever the capping group used.
  • PhAc-ALGP-Dox is cleaved by the enzyme into LGP-Dox (25% metabolite released after 3 hours of incubation at 37°C).
  • PhAc-AIGP-Dox is cleaved by CD10 into IGP-Dox (18% hydrolysis after 3 hours of incubation).
  • PhAc-DLGP-Dox and PhAc-GPGP-Dox, PhAc-KLGP-Dox and PhAc-TSGP-Dox are not or very slightly activated by CD10.
  • TOP hydrolyses Suc- ⁇ ALAL-Dox and PhAc-ALAL-Dox to release 64% and 44% of AL-Dox respectively. None of the ALAF-Dox and ALPF-Dox derivatives are cleaved by TOP.
  • TOP activates PhAc-ALGP-Dox, PhAc-KLGP-Dox and PhAc-TSGP-Dox into GP-Dox (72, 31 and 38 % of metabolite detected after 3 hours of incubation in the presence of the enzyme).
  • PhAc-DLGP-Dox, PhAc-GPGP-Dox PhAc-AIGP-Dox are not cleaved by TOP.
  • the sensitivity of the capped tetrapeptidic Dox prodrugs to TOP and CD10 is not contradictory with their blood stability. Most of the blood peptidases are exoproteases for which the capped tetrapeptidic Dox prodrugs are inaccessible as substrates. TOP and CD10 are, however, endoproteases unaffected by the presence of the capping group.
  • Suc- ⁇ ALAL-Dox, and PhAc-ALAL-Dox are activated by tumor cell secreted enzymes to release L-Dox and to a lesser extent AL-Dox and Dox.
  • PhAc-ALAF-Dox, PhAc-ALPF-Dox, and Suc- ⁇ ALPF-Dox are all activated into F-Dox by tumor cell secreted enzymes.
  • PhAc-ALGP-Dox is hydrolyzed in the presence of tumor cell conditioned medium into GP-Dox and Dox (56% and 24% of all metabolites detected after 5 hours, respectively), whereas mainly GP-Dox is detected after PhAc-KLGP-Dox and PhAc-TSGP-Dox activation.
  • PhAc-DLGP-Dox, PhAc-AIGP-Dox and PhAc-GPGP-Dox are not activated in the presence of LS174T tumor cells secreted peptidases.
  • PhAc-ALAL-Dox and PhAc-ALGP-Dox compounds are better cleaved by tumor cell secreted enzymes and have been selected for further in vivo analysis.
  • Cultured tumor cells are able to convert PhAc-ALGP-Dox to GP-Dox, LGP-Dox to Dox, and GP-Dox to Dox, which indicates the presence of CD10 and/or TOP (PhAc-ALGP-Dox to LGP-Dox or GP-Dox) at the one hand and of CD26 and/or FAP (GP-Dox to Dox) at the other hand.
  • This also confirms a two-step activation process of the prodrug occurring fully extracellularly, i.e., intracellular (lysosomal) processing of the prodrug is not required.
  • L-Dox released from Suc- ⁇ ALAL-Dox
  • CD26 sekunder-Dox
  • FAP prolyl peptidases
  • CD26 (1 ⁇ g/ml) or FAP did not hydrolyze either of PhAc-ALGP-Dox, LGP-Dox (efficiently released from PhAc-ALGP-Dox by TOP) or Suc- ⁇ ALAL-Dox.
  • GP-Dox was shown to be a good substrate for CD26 and FAP and was cleaved into Dox (97% conversion by CD26; 62% conversion by FAP).
  • PhAc-ALGP-Dox it is important to notice that in this way, the specificity of PhAc-ALGP-Dox to cancer cells is increased.
  • TOP is involved in the prodrug activation process of Suc- ⁇ ALAL-Dox or PhAc-ALAL-Dox (releasing AL-Dox) and of PhAc-ALGP-Dox (releasing GP-Dox).
  • AL-Dox is more generally sensitive to conversion into L-Dox and Dox by cell-secreted peptidases (and with L-Dox being automatically hydrolyzed into Dox intracellularly)
  • GP-Dox seems sensitive to peptidases predominantly released by tumor cells, such as CD26 and FAP.
  • PhAc-ALAL-Dox and PhAc-ALGP-Dox were dissolved in saline. Compounds were administered by intravenous bolus injection in the lateral tail vein of OF-1 mice (10 ⁇ l/g). The in vivo toxicity was evaluated by monitoring body weight.
  • Results in figure 1 show the high toxicity of PhAc-ALAL-Dox at 160 ⁇ mol/kg. No significant body weight loss is observed in the group treated with PhAc-ALAL-Dox at 80 ⁇ mol/kg.
  • PhAc-ALGP-Dox injection at the doses of 240 and 320 ⁇ mol/kg is well tolerated.
  • a moderate body weight loss with a maximum of 15% at day 28 is recorded in the PhAc-ALGP-Dox 240 ⁇ mol/kg treated group showing its lower toxicity in comparison with PhAc-ALAL-Dox.
  • Injection of PhAc-ALGP-Dox at 320 ⁇ mol/kg induced a significant body weight loss and one mouse was found dead at day 12.
  • LS 174T and MX-1 tumors were established by a subcutaneous implantation of cells (3x10 6 and 10 7 cells injected respectively) in the right flank of 6 weeks old female NMRI nude mice (Harlan). Treatments were initiated when the tumors had reached a size of 150-200 mm 3 (calculated using the following formula: [length x width 2 ]/2). The day of the first injection animals were randomly assigned to groups of 4 animals. Doxorubicin, and PhAc-ALGP-Dox were dissolved in saline. Compounds were delivered by bolus intravenous injection (i.v.) in the lateral tail vein at 10 ⁇ l/g. During the course of the experiment, clinical signs, body weight and tumor volume were controlled twice a week.
  • Results are presented as the evolution of mean tumor volume as a function of time.
  • Optimal T/C ratio of mean tumor volume of treated versus control mice
  • PhAc-ALGP-Dox was injected twice (once weekly) i.v. at 140 ⁇ mole/kg and 160 ⁇ mole/kg in nude mice bearing subcutaneously implanted LS174T tumors (colon carcinoma). Their body weight, and the tumor size were followed for 28 days and compared with the Dox (15 ⁇ mol/kg) and NaCl treated animals groups. Significant and similar antitumor activity was observed in all treated groups. (Table 4).
  • PhAc-ALGP-Dox PhAc-ALGP-Dox Doxorubicin Dose 140 ⁇ mol/kg 160 ⁇ mol/kg 15 ⁇ mol/kg TGI [% (day)] 79 (28) 81 (28) 55 (28)
  • PhAc-ALGP-Dox was injected four times i.v. (at day 0, 3, 6 and 9) at the dose of 100 ⁇ mol/kg in nude mice bearing subcutaneously implanted MX-1 tumors (mammary carcinoma). Their body weight and the tumor size were followed for 29 days and compared with the Dox (8 ⁇ mol/kg) and NaCl treated animals groups. No significant body weight loss and similar significant antitumor activity (inhibition of tumor growth >60%) were observed for the 2 tested drugs ( Figure 3 and Table 5). Table 5. Tumor Growth Inhibition. Mean RTV was calculated for each group at the end of the study.
  • % TGI percentage tumor growth inhibition
  • the hearts were homogenized with an Ultraturrax homogenizer in 1.5 mL water.
  • the protein concentration was measured using the microBCA protein assay (Pierce).
  • Doxorubicin and PhAc-ALGP-dox were injected i.v. bolus at the equimolar dose of 86.2 ⁇ mol/kg to wild type female OF-1 mice. Evolution of drug and metabolites concentration in plasma and cardiac tissue was determined by HPLC analysis. About 90% of the drug plasma concentration was eliminated in the first five minutes after injection of Dox or PhAc-ALGP-Dox ( Figure 4 A and B ). Less than 1% of the PhAc-ALGP-Dox was rapidly hydrolysed into LGP-Dox, GP-Dox and Dox. These metabolites were no longer detected after 1 hour.
  • the plasma area under curve (AUC) value for Doxorubicin after injection of Doxorubicin is 63 times higher than after injection of PhAc-ALGP-Dox (Table 6). Since the heart is the target for an important toxicity of Doxorubicin, the cardiac tissue concentration of the free drug was determined. Also determined were the heart AUCs for Doxorubicin after injection of Doxorubicin, and heart AUCs for Dox, GP-Dox and PhAc-ALGP-Dox after administration of PhAc-ALGP-Dox (Table 6). The Dox heart AUC after PhAc-ALGP-Dox administration is 25 times lower than after Dox administration at equimolar dose.
  • PhAc-ALGP-Dox would be significantly less cardiotoxic than Doxorubicin.
  • Table 6 Pharmacokinetic AUC values of PhAc-ALGP-Dox and of its metabolites vs Doxorubicin in cardiac tissue after one i.v. bolus injection to OF-1 mice at the dose of 86.2 ⁇ mol/kg AUC (area under curve)
  • Dox PhAc-ALGP-Dox Dox GP-Dox PhAc-ALGP-Dox Plasma ( ⁇ M.h) 63 1 1 1 97 Heart (pmol.h/mg protein) 5863 235 11 23
  • the in vitro cardiotoxicity test was carried out in a relevant and predictive in vitro model for cardiac safety screening in early lead optimization using mouse embryonic stem cell derived cardiomyocytes (Cor.At®, Axiogenesis (Germany)).
  • Cor.At® cardiomyocytes provide a standardized, homogenous and reproducible cell system for the in vitro classification of a compound's cardio-cytotoxic potential.
  • the neutral red uptake test was used to determine effects which directly affect the viability and integrity of cardiac cells when compared to a non-specific reference cell type, e.g. mouse fibroblasts (MEF).
  • MEF mouse fibroblasts
  • CorAt cardiomyocytes were incubated in the presence of increasing concentrations of PhAc-ALGP-Dox or of Doxorubicin. Cell viability was determined after 48 h using the neutral red uptake test. Mouse embryonic fibroblasts (MEF) were used as control cells to distinguish cardiac specific toxicity from general cytotoxicity.
  • the dose response curve of PhAc-ALGP-Dox ( Figure 5 ) did show a moderate toxic effect on Cor.At cardiomyocytes only at the highest concentration tested (20 ⁇ g/ml). At this concentration, the effect on MEF is less pronounced (81% viability vs. 37% viability). For the MEF, no IC50 is reached with this compound.
  • PhAc-ALGP-Dox did not show any toxic effect.
  • the dose response curve of Doxorubicin did show a severe toxic effect on Cor.At cardiomyocytes as well as on MEF at the two highest concentrations tested (20 ⁇ g/ml and 2 ⁇ g/ml).
  • the compound did show a moderate toxic effect on Cor.At cardiomyocytes, but only a marginal effect on MEF (67% viability of Cor.At cardiomyocytes vs. 89% viability of MEF).
  • the effect on Cor.At cardiomyocytes is only slightly higher than on MEF, the compound is considered to exert a cardiotoxic effect, which may be masked by a general cytotoxic effect.
  • EXAMPLE 8 Assessment of PhAc-ALGP-Dox activation at the tumor site after single iv bolus injection in nude mice bearing LoVo colon carcinoma xenograft
  • PhAc-ALGP-Dox The tumor activation of PhAc-ALGP-Dox was assessed using athymic mice (nude/nude NMRI) carrying ectopic xenograft of human LoVo colon carcinoma. LoVo tumors were established by a subcutaneous implantation of cells (10 7 cells) in the right flank of 6 weeks old female NMRI nude mice (Harlan). Drugs or controls were administered four weeks after subcutaneous implantation of the xenograft. On the day of injection, animals were randomly assigned to groups of 4 animals. The PhAc-ALGP-Dox conjugate was dissolved in saline at increasing doses (1.5, 3.5, 5, 10, 20, 30, 46, and 62 mM).
  • mice were sacrificed by cervical dislocation and tumors were collected, rinsed in phosphate buffer saline and homogenized. An extraction of drugs from tumor homogenates was performed with acetonitrile and Doxorubicin present in tumors was quantified by HPLC analysis.
  • Results in Figure 6 show that Dox tumor concentration increases with the injected dose of PhAc-ALGP-Dox to reach a plateau value at 200 ⁇ mol/kg. Results of this example indicate that a limited prodrug activation rate and availability at the tumor site could depend on the maximum of enzyme activity available during the duration of the contact with PhAc-ALGP-Dox.
  • PhAc-ALGP-Dox was dissolved in saline and administered by single or multiple intraperitoneal (ip) injections in the lateral tail vein of OF-1 mice (10 ⁇ l/g). PhAc-ALGP-Dox was administered at similar cumulative doses of 280 and 560 ⁇ mol/kg following different injections schedules: single ip injection; 5 consecutive daily ip injections at 56 and 112 ⁇ mol/kg or twice a day for five consecutive days at the doses of 28 and 56 ⁇ mol/kg. The in vivo toxicity was evaluated by monitoring the body weight.
  • PhAc-ALGP-doxorubicin is, in these conditions, about 15 times less toxic.
  • EXAMPLE 10 Pharmacokinetic and tissue quantification of PhAc-ALGP-Dox and its metabolites after single intraperitoneal injection in mice in comparison with Doxorubicin at equimolar dose
  • the hearts were homogenized with an Ultraturrax homogenizer in 1.5 mL water.
  • the protein concentration was measured using the microBCA protein assay (Pierce).
  • the blood concentration of Dox decreased rapidly within one hour to a very low concentration that remained stable up to 24hours after injection.
  • the AUC value for Doxorubicin was 70.3 ⁇ M.h.
  • the Doxorubicin cardiac tissue concentration was measured 24h after ip injection of PhAc-ALGP-Dox or Doxorubicin at equimolar dose. Results in Table 7 show that Doxorubicin accumulates 19 times less after intraperitoneal injection of 92 ⁇ mol/kg PhAc-ALGP-Dox than after injection of Doxorubicin at equimolar dose. Table 7. In vivo cardiac concentration of Dox after intraperitoneal injection of Dox and PhAc-ALGP-Dox at 92 ⁇ mol/kg. Mice were sacrificed 24 hours after drug administration and hearts were collected. Drug concentration was determined by HPLC analysis after extraction from tissue homogenates.
  • Results are expressed in pmol/mg protein ⁇ SD (concentrations of drugs and proteins were corrected taking into account of the blood remaining in the cardiac tissue). treatments Dox concentration in heart after 24h pmol/mg protein Dox 92 ⁇ mol/kg 253 ⁇ 60 PhAc-ALGP-Dox 92 ⁇ mol/kg 13 ⁇ 3
  • PhAc-ALGP-Dox was assessed using athymic mice (nude/nude NMRI) carrying ectopic xenograft of human LoVo colon carcinoma or of MX-1 mammary carcinoma in comparison with free Doxorubicin.
  • Tumors were established by a subcutaneous implantation of cells (10 7 cells) in the right flank of 6 weeks old female NMRI nude mice (Harlan). Treatments were administered when the tumors reached a size of 150-200 mm 3 (measured using a caliper and calculated with the following formula: [length x width 2 ]/2). Animals were randomly assigned to groups of 4 to-6 animals.
  • Doxorubicin and PhAc-ALGP-Dox were dissolved in saline.
  • mice bearing Lovo xenografts received twice a day (at 5-6 h interval) for 5 consecutive days (2Q1D5; total of 10 injections) intraperitoneal injections of saline, or of Doxorubicin at 0.5, 1 and 2 ⁇ mol/kg, or of Phac-ALGP-Dox at 25, 35 and 50 ⁇ mol/kg. Their body weight and the tumor size were followed and compared ( Figure 9 ). Tumor measurements were stopped in NaCl and Doxorubicin treated groups when tumor necrosis occurred. No significant body weight loss was recorded in this experiment. PhAc-ALGP-Dox induced a dose-dependent antitumor efficacy and increase in tumor growth delay (Table 8).
  • the 50 ⁇ mol/kg PhAc-ALGP-Dox treatment protocol resulted in a growth delay of 17 days whereas the highest dose of 2 ⁇ mol/kg Doxorubicin alone induced a growth delay of only 6 days.
  • the absolute growth delay induced by each treatment is calculated as the time in days for tumors in treated mice to grow from 190 to 1,000 mm 3 minus the time in days for tumors to reach the same size in vehicle-treated mice (TGD).
  • the absolute growth delay induced by each treatment was calculated as the time in days for tumors in treated mice to grow from 190 to 1,000 mm 3 minus the time in days for tumors to reach the same size in vehicle-treated mice (TGD).
  • MX-1 xenografted mice received at 72h interval 2 cycles of 1week with 2 daily ip injections of PhAc-ALGP-Dox at 50 ⁇ mol/kg or of Doxorubicin at 1 ⁇ mol/kg or 1.5 ⁇ mol/kg (2Q1D5 x2W; total of 20 injections).
  • Doxorubicin at 1.5 ⁇ mol/Kg was very toxic and induced severe body weight loss and death of animals. No significant body weight loss was recorded for Doxorubicin at 1 ⁇ mol/kg (MTD) and for 50 ⁇ mol/kg PhAc-ALGP-Dox.
  • Results in Figure 11 show that 50 ⁇ mol/kg PhAc-ALGP-Dox significantly inhibits tumor growth and has improved efficacy as compared with 1 ⁇ mol/kg Doxorubicin (MTD).
  • MTD Doxorubicin
  • PhAc-ALGP-Dox was tested in the well described B16-F10 lung metastatic melanoma model.
  • 5x10 7 B16-F10 murine melanoma cells were injected in the lateral vein of C57BL6 mice.
  • Treatments started three days after cells injection.
  • Animal received twice a day (at 5-6 h interval) for 5 consecutive days intraperitoneal injections (total 10 injections/mouse) of saline or of Doxorubicin at 2 and 3.5 ⁇ mol/kg or of PhAc-ALGP-Dox at 50 ⁇ mol/kg.
  • Five mice per group were sacrificed at day 14 after cells injection. Lungs were collected and processed for melanin quantification ( Molecular Pharmacology, 74: 1576-1586, 2008 ). Survival was determined by observation of the remaining mice.
  • PhAc-ALGP-Dox significantly inhibits the formation of lung metastasis and increases survival of mice as compared with NaCl and Doxorubicin treated groups ( Figure 12 ).
  • the amount of melanin in lung homogenate was 501 mg/mL in the control group, 183 and 53 mg/mL in the 2 and 3.5 ⁇ mol/kg Doxorubicin treated groups and 16 mg/mL in the 50 ⁇ mol/kg PhAc-ALGP-Dox treated group.
  • the median survival was 20, 24, 28 and 36 days in groups receiving NaCl, 2 and 3.5 ⁇ mol/kg Doxorubicin or 50 ⁇ mol/kg PhAc-ALGP-Dox, respectively.
  • HCT116 cells were subcutaneously injected into SCID mice. Once xenografts were established, they were excised and orthotopically implanted into the ceacum of other female ⁇ -irradiated SCID mice using microsurgical techniques. On day 12 after the cancer cells injection, the mice were randomized in four groups of 16. They received for 5 consecutive days 2 daily intraperitoneal injections of saline, of Doxorubicin at 2 ⁇ mol/kg and of PhAc-ALGP-Dox at 35 and 50 ⁇ mol/kg respectively. On day 34 after injection into the caecum of colonic cancer cells the animals were sacrificed, the number of metastases was counted macroscopically and the primary tumors weighted.
  • the control group gave a mean primary tumor weight of 0.88g with a SD of 0.41.
  • the second group at 30 ⁇ mol/kg of PhAc-ALGP-Dox had a primary tumor weight of 0.69 g with a SD of 0.25 while the group at 50 ⁇ mol/kg PhAc-ALGP-Dox presented a significant tumor weight loss with 0.44 g with a SD of 0.10.
  • the number of hepatic metastases were respectively of 20 (SD of 33) and of 24 (SD of 26) for the controls and the group treated at 30 ⁇ mol/kg PhAc-ALGP-Dox.
  • the effect of 50 ⁇ mol/kg PhAc-ALGP-Dox was very significant with a mean number of metastases of 1.78 with a SD of 2.9.
  • PhAc-ALGP-Dox was not totally devoid of toxicicty, this prodrug could at least be retained for treating/preventing metastases (e.g. hepatic metastases of colon carcinoma) if it would not be effective against the primary tumor itself (e.g. colon carcinoma) at non-toxic levels.
  • metastases e.g. hepatic metastases of colon carcinoma
  • Figure 13 A gives the mean and SD of the body weight variation of the two groups and 13 B the white blood cells variations as a percentage of WBC on day 0 for each mouse.
  • PhAc-ALGP-Dox Chronic cardiac toxicity of PhAc-ALGP-Dox in mice was morphologically evaluated as previously described by Bertazzoli et al. 1979 (Cancer Treat Rep 63, 1877-1883 ).
  • CD1 female white mice were treated by bolus intravenous injection in the tail vein at 10 ⁇ L/g (6 mice per group). Compounds were injected twice a week, ten times. Animals were not treated for 2 weeks between the first four injections and the last six injections to allow recovery of the bone marrow depression.
  • the treatment dose-levels of PhAc-ALGP-Dox were: 13.8; 27.6; 55.2; and 82.8 ⁇ mol/kg. Doxorubicin 6.9 ⁇ mol/kg was used as a reference.
  • mice were deeply anaesthetized by intraperitoneal injection of nembutal (50 mg/kg) and exsanguinated.
  • the hearts were carefully collected, rinsed in NaCl 9 ⁇ and fixed in a 10% formaldehyde solution. Samples were processed for histopathological analysis (CITox Lab, France). The heart was trimmed, embedded in paraffin wax, sectioned at a thickness of 4 microns and stained with hematoxylin-eosin before microscopic evaluation.
  • body weight was controlled before each injection or once a week.
  • Atrial myofiber atrophy and inflammatory infiltrate were seen in 4 mice out of 5, along with fibrin thrombi in 3 mice out of 5. In one of these mice, there was also degeneration/necrosis of myofibers.
  • the administration of PhAc-ALGP-Dox did not induce any pathologic microscopic findings in the heart at any dose levels (i.e. up to a 12 times higher dose as compared with Doxorubicin). Table 11.
  • PhAc-ALGP-Dox chronic cardiotoxicity in mice in comparison with Doxorubicin Treatment
  • NaCl Doxorubicin PhAc-ALGP-Dox Dose ( ⁇ mol/kg) 0 6.9 13.8 27.6 55.2 82.8 Number of mice 6 5 6 6 6 6 Vacuolation; myofib.
  • PhAc-ALGP-Dox does not induce leukopenia at active doses and does not present cumulative cardiotoxicity in a mouse model at a dose equivalent to 12 times that of Doxorubicin at its MTD level.
  • Maytansine is a potent microtubule-targeted compound that induces mitotic arrest and kills tumor cells at sub-nanomolar concentrations. However, its side effects and lack of tumor specificity have prevented successful clinical use. It inhibits microtubule assembly, inducing microtubule disassembly, and disrupts mitosis. Maytansine exhibits cytotoxicity against many tumor cell lines and displays about 100-fold higher cytotoxicity than the Vinca alkaloids and about 1000-fold higher toxicity than doxorubicin.
  • Maytansine can be conjugated to PhAc-ALGP via a self-immolating spacer reacting with its free -NH or OH group. Maytansine is commercially available (e.g. Medkoo Biosciences; Xuzkou Kaiyide Chemical Co).
  • Geldanamycin and derivates thereof are a family of a benzoquinone ansamycins, antibiotics originally isolated on the basis of their weak antibiotic activity that were subsequently shown to display potent antitumor activity.
  • Geldanamycin induces, compared to their normal cellular counterparts, preferential degradation of proteins that are mutated in tumor cells such as v-src, bcr-abl and p53. This effect is mediated via Hsp90.
  • geldanamycin has several major disadvantages as antitumor agent which has led to the development of geldanamycin analogues, in particular analogues containing a substitution on the 17 position.
  • geldanamycin derivatization of geldanamycin at the position leads to 17AAG (17-(allylamino)-17-demethoxygeldanamycin) hat has lower in vivo toxicity than geldanamycin. Even though Hsp90 affinity to 17AAG is less than to geldanamycin, 17AAG and geldanamycin gave biologic effects in malignant cells at similar or same concentrations.
  • Geldanamycin binds with high affinity to the ATP binding pocket of Hsp90.
  • Hsp90 is a ubiquitous molecular chaperone critical for the folding, assembly and activity of multiple mutated and overexpressed signaling proteins that promote the growth and/or survival of tumor cells. Binding of geldanamycin to Hsp90 causes the destabilization and degradation of its target.
  • Taxanes are diterpenes produced by the Taxus plants. They include paclitaxel (Taxol) and docetaxel (taxotere; see Figure below)
  • Paclitaxel is one of several cytoskeletal drugs that target tubulin. Paclitaxel-treated cells have defects in mitotic spindle assembly, chromosome segregation, and cell division. Paclitaxel stabilizes the microtubule polymer, protecting it from depolymerisation, and thereby blocks mitosis. Recent studies have demonstrated that suppression of dynamics occurs at concentrations lower than those needed to block mitosis. At the higher therapeutic concentrations, paclitaxel appears to suppress microtubule detachment from centrosomes, a process normally activated during mitosis. Paclitaxel is approved for treatment of ovarian, breast and lung cancers and Kaposi's sarcoma.
  • Docetaxel or taxotere differs from paclitaxel at two positions in its chemical structure. It has a hydroxyl functional group on carbon 10 (where paclitaxel has an acetate ester), and a tert-butyl carbamate ester exists on the phenylpropionate side chain instead of the benzylamide in paclitaxel.
  • the carbon 10 functional group change causes docetaxel to be more water soluble than paclitaxel.
  • the hydroxyl group on carbon 2 remains unmodified.
  • Paclitaxel was linked to antibodies by a simple reaction ( Guillemard & Saragovi 2001; Cancer Res 61, 694-699 ). Paclitaxel was derivatized by reacting glutaric aldehyde to give 2'-glutaryl-paclitaxel containing a cleavable ester bond. 2'-glutaryl-paclitaxel was then activated by removal of a hydroxyl group with carbodiimide and bound to an antibody directly via its amino-group to form a peptide linkage. This technique was used recently ( Garcia et al Oncogene 2012; doi: 10.1038/onc.2012.283 ) to link taxol to an antiherceptin monoclonal antibody. Experimental results indicate that the conjugate is active in experimental tumors indicating that the drug is released in vivo.
  • Paclitaxel was also conjugated to antibodies after succinylation of paclitaxel at the 2'position and coupling to antibodies via an amide bound ( Safavy et al. 2003; Bioconj Chem 14, 302-310 ). Similar methods of conjugating activated 2'-glutaryl or 2'-succinyl paclitaxel to the PhAc-ALGP- tetrapeptide ; alternatively a self-immolating spacer linked on the carboxyl group of the succinyl-paclitaxel is used.And it is reasonable to expect release in vivo of taxol from the PhAc-ALGP-paclitaxel conjugate, certainly taking into account that a tetrapeptide exerts a much smaller steric hindrance than antibodies do.
  • Camptothecin (CPT; structure depicted below) is a cytotoxic quinoline alkaloid that inhibits the DNA enzyme topoisomerase I (Topol). CPT showed remarkable anticancer activity in preliminary clinical trials but suffers from low solubility and (high) adverse drug reaction. Because of these disadvantages synthetic and medicinal chemists have developed numerous syntheses of camptothecin and various derivatives. Two CPT analogues have been approved and are used in cancer chemotherapy today: topotecan and irinotecan.
  • substitution at position 7, 9, 10 and 11 can have positive effect on CPT activity and physical properties, e.g. potency and metabolic stability. Enlargement of the lactone ring by one methylene unit also enhances its abilities, as in homocamptothecin. Substitution at position 12 and 14 leads to an inactive derivative.
  • Vinblastine (structure depicted below) is an anti-microtubule drug used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-small cell lung cancer, breast cancer, head and neck cancer, and testicular cancer. It is also used to treat Langerhans cell histiocytosis. Vinblastine was traditionally obtained from Catharanthus roseus, also known as Vinca rosea, a Madagascar periwinkle. It is generated in the plant by the joining of the alkaloids catharanthine and vindoline.
  • Vincristine is a close analog differing from vinblastine only by CHO instead of CH3 on N 1 . Although very similar to vinblastine in structure it has other therapeutic indications and a very severe side effect. Its main indications are non-Hodgkin's lymphoma, in acute lymphoblastic leukemia, and in treatment for nephroblastoma (Wilms' tumor, a kidney tumor most common in young children). The main side-effects of vincristine are peripheral neuropathy, hyponatremia, constipation, and hair loss. Peripheral neuropathy can be severe, and be a reason to avoid, reduce, or stop the use of vincristine.
  • PhAc-ALGP-vinblastine or -vincristine conjugates can be obtained by linking a desacetyl vinblastine or -vincristine via a self-immolative spacer bound on their carbon C 4 .
  • Kandukuri et al. 1985 J Med Chem 28, 1079-1088 ) developed a synthesis method of amino acid derivatives of vinblastine involving an amide linkage with the carboxylic end side chain of the amino acid.
  • the linkage was obtained by a mixed anhydride condensation between the C 4 deacetylvinblastine and N-maleoyl amino acids; vinblastine-C4 amino acid maleoyls were also conjugated to lactosaminated serum albumin and shown to be active against HepG2 carcinoma ( Rao et al. 1989; Anticancer Res 9, 973-979 ).
  • Logically the same procedure is applicable to vincristine. Conjugation of vinblastine or vincristine to PhAc-ALGP is likewise achievable with this method.
  • Vinblastine is commercially available (e.g. Medkoo Biosciences), as well as vincristine (e.g. Tocris Bioscience, Medkoo Biosciences).
  • Methotrexate is an antimetabolite and antifolate drug. It is used in treatment of cancer and of autoimmune diseases.
  • Methotrexate was originally developed and continues to be used for chemotherapy either alone or in combination with other agents. It is effective for the treatment of a number of cancers including: breast, head and neck, leukemia, lymphoma, lung, osteosarcoma, bladder, and trophoblastic neoplasms. The most common adverse effects include: ulcerative stomatitis, low white blood cell count and thus predisposition to infection, nausea, abdominal pain, fatigue, fever, dizziness, and acute pneumonitis.
  • Methotrexate is thought to affect cancer and rheumatoid arthritis by two different pathways.
  • methotrexate allosterically inhibits dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis.
  • DHFR dihydrofolate reductase
  • the affinity of methotrexate for DHFR is about one 1000-fold that of folate.
  • DHFR catalyses the conversion of dihydrofolate to the active tetrahydrofolate. Methotrexate, therefore, inhibits the synthesis of DNA, RNA, thymidylates, and proteins.
  • DHFR For the treatment of rheumatoid arthritis, inhibition of DHFR is not thought to be the main mechanism, but rather the inhibition of enzymes involved in purine metabolism, leading to accumulation of adenosine, or the inhibition of T cell activation and suppression of intercellular adhesion molecule expression by T cells.
  • Umemoto et al. 1989 (Int J Cancer 43, 677-684 ) described a method to link methotrexate via free carboxyl group to antibodies with a of Ala-Leu-Ala-Leu linker This method can likewise be applied to link methotrexate to PhAcALGP peptide and will restore the free carboxyl group after enzymatic cleavage.
  • Derivatives of the alpha-carboxylate group are relatively non-active and non-toxic in vitro since a free alpha-carboxylate group is necessary for the binding of methotrexate to DHFR
  • methotrexate Potential prodrugs of methotrexate were also produced in which the 2-aminogroup was acylated with alpha-amino acids ( Smal et al. 1995; Biochemical Pharmacology 49, 567-574 ).. These aminoacyl derivatives are substituted at the 2-NH 2 pteridine ring of methotrexate. Importantly, the 2-leucyl-methotrexate derivative is rapidly cleaved and activated in presence of serum illustrating its sensitivity to serum exoproteases. This makes it plausible for a PhAc-ALGP-methotrexate conjugate to display tumor cell specific anticancer activity. Linking methotrexate to PhAc-ALGP is performed via the above-described technique. Methotrexate is commercially available (e.g. CF Pharma Ltd, Yaskika Pharmaceuticals).
  • Aminopterin (4-aminopteroic acid), a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties.
  • Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase. Its structure is very similar to that of methotrexate and it has also a 2-NH 2 on its pteridine moiety. Developed before methotrexate, it was superseded by the latter early in the 1950's because of its greater toxicity that could result from a greater activity. Greater effectiveness was confirmed recently in the treatment of acute leukemia ( Cole et al. 2005; Clin Cancer Res 11, 8089-8096 ).
  • PhAc-ALGP-aminopterin conjugate can be synthesized by the methods described for PhAc-ALGP-methotrexate and enhanced specificity of the anticancer activity for tumor cells is likewise plausible.
  • Aminopterin is commercially available (e.g. Cameo Chemicals, Sigma Aldrich).
  • Amrubicin (structure depicted below) is a third-generation, synthetic anthracycline analogue that has demonstrated substantial clinical efficacy in the treatment of small cell lung cancer.
  • Amrubicin is a potent topoisomerase II inhibitor and is being studied as a single agent and in combination with anti-cancer therapies for a variety of solid tumors, including lung and breast. It has been granted the orphan drug classification by the FDA.
  • PhAc-ALGP conjugation of PhAc-ALGP to this anthracycline is applied.
  • the presence of the PhAc-ALGP increases tumor cell selectivity of the anticancer activity of amrubicin.
  • Amrubicin is commercially available (e.g. Medkoo Biosciences, Santa Cruz Biotech).
  • a GP-dipeptide is conjugated to the cytotoxic compound and analytical methodology is developed for detecting and/or quantifying the GP-cytotoxic compound conjugate, the cytotoxic compound, and intermediates between the two.
  • analytical methodology may include one or more of spectrophotometry, high performance liquid chromatography (HPLC), mass spectrometry (MS), combined HPLC and MS, NMR and MALDI-TOF (matrix-assisted laser desorption/ionization - Time-of-flight), or even UPLC-MS/MS (ultra-high performance liquid chromatography with tandem mass-spectrometry).
  • the purified GP-cytotoxic compound conjugate is tested in vitro in a biological system to confirm its cytotoxicity.
  • GP-cytotoxic compound conjugates confirmed to be cytotoxic synthesis of the complete PhAc-ALGP-cytotoxic compound conjugate is performed and analytical methodology is developed for detecting and/or quantifying the PhAc-ALGP-cytoyoxic compound conjugate, the cytotoxic compound, and intermediates between the two.
  • the cytotoxic activity of the starting compound (unconjugated, the "parent” drug) and of its conjugated derivative (the "GP-drug”) will be tested on in vitro cell lines.
  • the cell lines will be at least some of those mentioned in the referenced publications mentioned hereinabove for each of the cytotoxic compounds. It will be necessary to do tests as a function of drug concentration and time of incubation.
  • the GP-drug should be much less active than the parent drug after short incubation times. With increasing incubation time, this difference could become less significant due to increased hydrolysis of the GP-drug by exoproteases present in serum that is part of the incubation media. In such case, the presence of exoproteases in serum could be confirmed, if possible, by incubation of the GP-drug in serum-free incubation media.
  • Another possible solution would be to intercalate a self-immolating spacer that restores the original drug after hydrolysis of the drug-spacer and proline bound.
  • One possible spacer of this type is PABC or PAB (para-aminobenzyloxycarbonyl), attaching the drug moiety to the ligand in the conjugate.
  • the linker moiety comprises a peptide sequence that is a substrate for an extracellular enzyme, for example FAP, that cleaves the peptide at an amide bond.
  • the peptide further contains a self-immolating moiety which connects the drug and the protein peptide sequence. Upon cleavage of the peptide sequence by an intracellular enzyme the self-immolating moiety cleaves itself from the drug moiety such that the drug moiety is in an underivatized and active form.
  • An alternative experiment consists of incubating the PhAc-ALGP prodrug with purified CD10 and TOP, and analyze conversion to GP-prodrug; after simultaneous incubation of the PhAc-ALGP prodrug with CD10, TOP, FAP and DPIV, the extent of conversion of PhAc-ALGP prodrug to free drug can be analyzed. Significantly high levels of conversion to GP-prodrug and free drug, respectively, are indicative of cytotoxic efficacy of the PhAc-ALGP prodrug in a cellular/tumor environment.
  • In vivo testing first determines the MTD of the prodrug conjugate by measuring the weight loss of mice injected with increasing doses of the prodrug. This is compared with the MTD of the free cytotoxic drug. The MTD will be determined as the dose not inducing a weight loss exceeding 20% of the original weight of the animals.
  • the prodrug and the free drug will be administered IV 2 times with a weekly interval, possibly repeated one or more times.
  • the doxorubicin prodrug in vivo activation of the prodrug could be very (s)low.
  • the MTD is determined in normal mice and in mice xenografted with a human tumor, after twice daily IP injections for 2 times 5 days.
  • slow infusion of the prodrugs is possible using osmotic or other programmable minipumps.
  • One or two different human xenograft tumor models are selected on the basis of published data obtained with the unconjugated/free cytotoxic compound. Prodrug and free drug are injected IP at the MTD determined as described above. Body weight and tumor volume are measured every two days and compared with results obtained with the free starting drug at its MTD.
  • pharmacokinetic tissue distribution studies of drug and prodrug may require adequate labeling of drug and prodrug too yield sufficient sensitivity in chemical analytical determination methods.
  • One type of labeling is radiolabeling, the compounds could e.g. be radiolabeled by tritium exchange or by neosynthesis with C -14 labeled precursors. Labeling should be such that the label, or a label is maintained in metabolites of the prodrug. Plasma pharmacokinetics are explored as well as tumor accumulation of the conjugated drug and its metabolites, combined with organ and tissue distribution.
  • mice were respectively treated with saline (control group), treated with doxorubicin (doxorubicin-treated group) and treated with PhAc-ALGP-dox (PhAc-ALGP-dox-treated group)
  • doxorubicin-treated group treated with PhAc-ALGP-dox
  • tumors treated with PhAc-ALGP-dox revealed stabilization of tumor volume at 105% ( Figure 16 ).
  • PhAc-ALGP-dox may in part be explained by the 7-d continuous administration of the drug via a minipump.
  • a second and more interesting explanation resides in a probably much higher release of doxorubicin in the tumor stroma than after administration of free doxorubicin (which can only be administered at a much lower dose).
  • the cytostatic and cytotoxic effect of the released doxorubicin on stromal cells may in turn strongly affect the growth of the cancerous cells.
  • This stromal effect may also explain the stabilizing effect on the tumor volume for 21 days without a reduction in their volume. Extended observation beyond 21 days, possibly in combination with repeated administration, using for example different vehicles with effect on active drug release, could synergistically result in a reduced tumor volume (after the destruction of all tumoral stromal cells).
  • mice body weight and general well-being were monitored.
  • No major side effects were observed and in general the animals' body weight did not drop below the acceptable value (20% of the starting body weight loss during the treatment).
  • One mouse was sacrificed for ethical reasons on day 13 in the PhAc-ALGP-dox group (body weight 79.8%, animal getting skinny from day 11, limited intraperitoneal ascites fluid found during the necropsy probably due to infection as consequence of the surgical implantation of the minipump).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Nanotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medical Informatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Genetics & Genomics (AREA)

Claims (12)

  1. Ein Prodrug mit der allgemeinen Struktur:

            [Cx-OP]y-D,

    wobei
    C eine Capping-Gruppe ist;
    OP ein Tetrapeptid mit der Sequenz ALGP (SEQ ID Nr. 1), TSGP (SEQ ID Nr. 4) oder KLGP (SEQ ID Nr. 6) ist;
    D ein Arzneistoff ist;
    x eine Ganzzahl ist, die mindestens 1 ist, wenn y = 1;
    y eine Ganzzahl ist, die mindestens 1 ist, wenn y größer als 1 ist, dann trägt mindestens 1 OP eine Capping-Gruppe; und
    wobei die Bindung zwischen C und OP und die Bindung zwischen OP und D direkt oder über eine Linker- oder Spacer-Gruppe erfolgt, und wobei, wenn y größer als 1 ist, die multiplen OP-Einheiten einzeln direkt oder über eine Linker- oder Spacer-Gruppe aneinander gebunden sind und/oder einzeln direkt oder über eine Linker- oder Spacer-Gruppe an D gebunden sind;
    oder ein pharmazeutisch akzeptables Salz davon.
  2. Das Prodrug oder dessen Salz gemäß Anspruch 1, wobei die genannte Capping-Gruppe C eine Phosphonoacetyl-Gruppe ist.
  3. Das Prodrug oder dessen Salz gemäß Anspruch 1, wobei der genannte Arzneistoff D ausgewählt ist aus Doxorubicin, Daunorubicin, Amrubicin, Vinblastin, Vincristin, Calicheamicin, Etoposid, Etoposidphosphat, CC-1065, Duocarmycin, KW-2189, Methotrexat, Methopterin, Aminopterin, Dichlormethotrexat, Docetaxel, Paclitaxel, Epithiolon, Combretastatin, Combretastatin-A4-Phosphat, Dolastatin 10, Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin, Mitomycin C, Porfiromycin, 5-Fluoruracil, 6-Mercaptopurin, Fludarabin, Tamoxifen, Cytosin-Arabinosid, Adenosin-Arabinosid, Colchicin, Halichondron B, Cisplatin, Carboplatin, Mitomycin C, Bleomycin, Melphalan, Chloroquin, Cyclosporin A und Maytansin.
  4. Das Prodrug oder dessen Salz gemäß den Ansprüchen 1 oder 3, wobei der genannte Arzneistoff D pegyliert ist.
  5. Das Prodrug oder dessen Salz gemäß einem der Ansprüche 1 bis 4, wobei das genannte Prodrug Compound I ist:
    Figure imgb0029
  6. Eine Zusammensetzung, welche das Prodrug oder dessen Salz gemäß einem der Ansprüche 1 bis 5 umfasst.
  7. Das Prodrug oder dessen Salz gemäß einem der Ansprüche 1 bis 5 oder die Zusammensetzung gemäß Anspruch 6 zur Verwendung bei der Behandlung von Krebs.
  8. Eine Methode zur Produktion des Prodrugs gemäß einem der Ansprüche 1 bis 5, wobei die genannte Methode folgende Schritte umfasst:
    (i) Beschaffen des Arzneistoffs;
    (ii) Binden des Arzneistoffs an eine gecappte Oligopeptid-Einheit, was im Prodrug resultiert; oder alternativ
    (ii') Binden des Arzneistoffs an eine Oligopeptid-Einheit, gefolgt von der Bindung der Capping-Gruppe an die Oligopeptid-Einheit, was im Prodrug resultiert; und
    (iii) Aufreinigen des in Schritt (ii) oder (ii') erhaltenen Prodrugs;
    wobei die Oligopeptid-Einheit ein Tetrapeptid mit der Sequenz ALGP (SEQ ID Nr. 1), TSGP (SEQ ID Nr. 4) oder KLGP (SEQ ID Nr. 6) ist;
  9. Eine Methode des Screenings von Prodrug-Kandidaten mit der allgemeinen Struktur

            [Cx-OP]y-D,

    wobei
    C eine Capping-Gruppe ist;
    OP ein Tetrapeptid mit der Sequenz ALGP (SEQ ID Nr. 1), TSGP (SEQ ID Nr. 4) oder KLGP (SEQ ID Nr. 6) ist;
    D ein Arzneistoff ist;
    x eine Ganzzahl ist, die mindestens 1 ist, wenn y = 1;
    y eine Ganzzahl ist, die mindestens 1 ist, wenn y größer als 1 ist, dann trägt mindestens 1 OP eine Capping-Gruppe; und
    wobei die Bindung zwischen C und OP und die Bindung zwischen OP und D direkt oder über eine Linker- oder Spacer-Gruppe erfolgt, und wobei, wenn y größer als 1 ist, die multiplen OP-Einheiten einzeln direkt oder über eine Linker- oder Spacer-Gruppe aneinander gebunden sind und/oder einzeln direkt oder über eine Linker- oder Spacer-Gruppe an D gebunden sind;
    wobei die genannte Methode folgende Schritte umfasst:
    (i) Beschaffen des Arzneistoffs D;
    (ii) Konjugation des Arzneistoffs D an [Cx-OP]y, um ein [Cx-OP]y-D-Prodrug zu erhalten;
    (iii) Kontaktieren des Arzneistoffs D und des Prodrugs [Cx-OP]y-D jeweils unabhängig mit in-vitro kultivierten Zellen;
    (iv) Bestimmen der Zytotoxizität des Arzneistoffs D und des Prodrugs [Cx-OP]y-D;
    (v) Identifizieren aus (iv) ein Prodrug [Cx-OP]y-D mit vergleichbarer zytotoxischer Aktivität wie Arzneistoff D; und
    (vi) Auswählen des in Schritt (v) identifizierten [Cx-OP]y-D als Prodrug-Kandidat.
  10. Eine Methode des Screenings von Prodrug-Kandidaten mit der allgemeinen Struktur

            [Cx-OP]y-D,

    wobei
    C eine Capping-Gruppe ist;
    OP ein Tetrapeptid mit der Sequenz ALGP (SEQ ID Nr. 1), TSGP (SEQ ID Nr. 4) oder KLGP (SEQ ID Nr. 6) ist;
    D ein Arzneistoff ist;
    x eine Ganzzahl ist, die mindestens 1 ist, wenn y = 1;
    y eine Ganzzahl ist, die mindestens 1 ist, wenn y größer als 1 ist, dann trägt mindestens 1 OP eine Capping-Gruppe; und
    wobei die Bindung zwischen C und OP und die Bindung zwischen OP und D direkt oder über eine Linker- oder Spacer-Gruppe erfolgt, und wobei, wenn y größer als 1 ist, die multiplen OP-Einheiten einzeln direkt oder über eine Linker- oder Spacer-Gruppe aneinander gebunden sind und/oder einzeln direkt oder über eine Linker- oder Spacer-Gruppe an D gebunden sind;
    wobei die genannte Methode folgende Schritte umfasst:
    (i) Beschaffen des Arzneistoffs D;
    (ii) Konjugation des Arzneistoffs D an [Cx-OP]y, um ein [Cx-OP]y-D-Prodrug zu erhalten;
    (iii) Kontaktieren von Prodrug [Cx-OP]y-D für 5 h bei 37 °C mit in-vitro kultivierten Zellen oder für 3 h bei 37 °C mit isolierten CD10- und/oder TOP-Peptidasen und mit isolierten FAP- und/oder DPIV-Peptidasen;
    (iv) Bestimmen der Konversion des Prodrugs [Cx-OP]y-D in freien Arzneistoff D;
    (v) Identifizieren aus (iv) ein Prodrug [Cx-OP]y-D, welches zu mindestens 50 % in D konvertiert wird; und
    (vi) Auswählen des in Schritt (v) identifizierten [Cx-OP]y-D als Prodrug-Kandidat.
  11. Die Methode gemäß Anspruch 9 oder 10, wobei der genannte Arzneistoff D ausgewählt ist aus Doxorubicin, Daunorubicin, Amrubicin, Vinblastin, Vincristin, Calicheamicin, Etoposid, Etoposidphosphat, CC-1065, Duocarmycin, KW-2189, Methotrexat, Methopterin, Aminopterin, Dichlormethotrexat, Docetaxel, Paclitaxel, Epithiolon, Combretastatin, Combretastatin-A4-Phosphat, Dolastatin 10, Dolastatin 11, Dolastatin 15, Topotecan, Camptothecin, Mitomycin C, Porfiromycin, 5-Fluoruracil, 6-Mercaptopurin, Fludarabin, Tamoxifen, Cytosin-Arabinosid, Adenosin-Arabinosid, Colchicin, Halichondron B, Cisplatin, Carboplatin, Mitomycin C, Bleomycin, Melphalan, Chloroquin, Cyclosporin A und Maytansin.
  12. Ein Kit bestehend aus einem Behälter, der das Prodrug oder dessen Salz gemäß einem der Ansprüche 1 bis 5 oder die Zusammensetzung gemäß Anspruch 6 enthält.
EP13820778.2A 2012-12-28 2013-12-27 Minimal toxische prodrugs Active EP2938360B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PL13820778T PL2938360T3 (pl) 2012-12-28 2013-12-27 Minimalnie toksyczne proleki

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261746621P 2012-12-28 2012-12-28
PCT/EP2013/078034 WO2014102312A2 (en) 2012-12-28 2013-12-27 Minimally toxic prodrugs

Publications (2)

Publication Number Publication Date
EP2938360A2 EP2938360A2 (de) 2015-11-04
EP2938360B1 true EP2938360B1 (de) 2019-09-11

Family

ID=49955306

Family Applications (1)

Application Number Title Priority Date Filing Date
EP13820778.2A Active EP2938360B1 (de) 2012-12-28 2013-12-27 Minimal toxische prodrugs

Country Status (12)

Country Link
US (4) US10076576B2 (de)
EP (1) EP2938360B1 (de)
JP (3) JP6523965B2 (de)
CN (2) CN104884091B (de)
AU (1) AU2013369261B2 (de)
CA (1) CA2896337C (de)
DK (1) DK2938360T3 (de)
EA (1) EA034046B1 (de)
ES (1) ES2759999T3 (de)
HK (1) HK1210043A1 (de)
PL (1) PL2938360T3 (de)
WO (1) WO2014102312A2 (de)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL2938360T3 (pl) 2012-12-28 2020-05-18 Cobiores Nv Minimalnie toksyczne proleki
US20140364368A1 (en) * 2013-06-06 2014-12-11 Massachusetts Institute Of Technology Stimulus responsive nanocomplexes and methods of use thereof
EP4299134A3 (de) 2014-06-13 2024-03-27 Bach Biosciences, LLC Fap-aktivierte therapeutische mittel und damit verbundene verwendungen
US9737556B2 (en) 2014-06-13 2017-08-22 Trustees Of Tufts College FAP-activated therapeutic agents, and uses related thereto
JP7224583B2 (ja) 2018-01-29 2023-02-20 日本メナード化粧品株式会社 ガレクチン－９産生促進剤
CN109456390B (zh) * 2018-12-27 2021-11-16 西华师范大学 一种人工合成多肽h-473及其应用
WO2022043256A1 (en) 2020-08-23 2022-03-03 Cobiores Nv Synergistic combinations of anticancer drugs linked to a tetrapeptidic moiety and immunotherapeutic agents
AU2021405744A1 (en) 2020-12-22 2023-08-03 Cobiores Nv Compounds comprising a tetrapeptidic moiety
CN112940248B (zh) * 2021-02-04 2023-07-18 西安交通大学 一种pH响应型金属配位聚前药纳米粒子及其制备方法
WO2022167664A1 (en) 2021-02-07 2022-08-11 Cobiores Nv Compounds comprising a tetrapeptidic moiety

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5767242A (en) 1994-04-20 1998-06-16 Boehringer Ingelheim Int'l Gmbh Isolated dimeric fibroblast activation protein alpha, and uses thereof
US5543396A (en) * 1994-04-28 1996-08-06 Georgia Tech Research Corp. Proline phosphonate derivatives
US7425541B2 (en) 1998-12-11 2008-09-16 Medarex, Inc. Enzyme-cleavable prodrug compounds
DE69942128D1 (de) * 1998-12-11 2010-04-22 Coulter Pharm Inc Prodrugs und verfahren zu deren herstellung
US6613879B1 (en) * 1999-05-14 2003-09-02 Boehringer Ingelheim Pharma Kg FAP-activated anti-tumour compounds
CA2391534A1 (en) 1999-11-15 2001-05-25 Drug Innovation & Design, Inc. Selective cellular targeting: multifunctional delivery vehicles
GB0027552D0 (en) * 2000-11-10 2000-12-27 Boehringer Ingelheim Pharma Anti-tumor compounds
EP1333033A1 (de) * 2002-01-30 2003-08-06 Boehringer Ingelheim Pharma GmbH & Co.KG Mittels FAP aktivierbare anti-tumoröse Verbindungen
US20060281897A1 (en) * 2003-08-22 2006-12-14 Andre Trouet Potentialization of the activation of high molecular weight prodrugs
US7399869B2 (en) 2005-05-19 2008-07-15 Genentech, Inc. Fibroblast activation protein inhibitor compounds and methods
WO2008015597A1 (en) * 2006-08-04 2008-02-07 Firmenich Sa Keto-esters as flavoring ingredients
ITTO20060852A1 (it) * 2006-11-30 2008-06-01 Univ Degli Studi Torino Peptidi metastasi-specifici e loro applicazioni diagnostiche e terapeutiche
US20100184706A1 (en) 2007-03-20 2010-07-22 Bachovchin William W Fap-activated chemotherapeutic compounds, and methods of use thereof
EP1977765A1 (de) 2007-04-03 2008-10-08 Diatos Peptid-Prodrugs
CA2687535C (en) * 2007-05-18 2017-08-22 Coley Pharmaceutical Gmbh Phosphate-modified oligonucleotide analogs with enhanced immunostimulatory activity
PL2938360T3 (pl) 2012-12-28 2020-05-18 Cobiores Nv Minimalnie toksyczne proleki

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
CN104884091A (zh) 2015-09-02
WO2014102312A2 (en) 2014-07-03
AU2013369261A1 (en) 2015-06-11
EP2938360A2 (de) 2015-11-04
HK1210043A1 (en) 2016-04-15
CN113476616A (zh) 2021-10-08
DK2938360T3 (da) 2019-11-25
US20160058880A1 (en) 2016-03-03
US10076576B2 (en) 2018-09-18
US20240123074A1 (en) 2024-04-18
PL2938360T3 (pl) 2020-05-18
ES2759999T3 (es) 2020-05-12
US20190175740A1 (en) 2019-06-13
US20200215196A1 (en) 2020-07-09
CA2896337A1 (en) 2014-07-03
JP2019055966A (ja) 2019-04-11
CA2896337C (en) 2022-08-23
JP2021073242A (ja) 2021-05-13
JP2016509586A (ja) 2016-03-31
JP6523965B2 (ja) 2019-06-05
WO2014102312A3 (en) 2014-08-21
EA034046B1 (ru) 2019-12-23
US10583194B2 (en) 2020-03-10
CN104884091B (zh) 2021-07-20
AU2013369261B2 (en) 2018-08-09
EA201590863A1 (ru) 2015-11-30

Similar Documents

Publication Publication Date Title
US20240123074A1 (en) Minimally toxic prodrugs
EP1404356B1 (de) Methode für die ausarbeitung von cd10-aktivierten prodrug-verbindungen
EP1144011B1 (de) Prodrugs und verfahren zu deren herstellung
AU2002316539A1 (en) CD10-activated prodrug compounds
JPH10508291A (ja) 化合物、製剤用組成物及びこれらを含む診断装置とこれらの利用
KR20200088402A (ko) 카텝신 b의 엑소펩티다제 활성에 의한 선택적인 절단의 기질로서 리간드-약물-접합체
KR102436012B1 (ko) 항암제 프로드러그 컨쥬게이트의 새로운 용도
US20240299562A1 (en) Compounds comprising a tetrapeptidic moiety
WO2022167664A1 (en) Compounds comprising a tetrapeptidic moiety
LOPEZ RIVAS Synthesis of integrin-targeting pro-drugs for the selective release of anti-tumor agents
Agustí Novel Cryptophycin Analogues and Conjugates for Tumor Targeted Therapy

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20150717

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20170428

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: COBIORES NV

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 31/395 20060101ALI20190219BHEP

Ipc: A61P 35/00 20060101ALI20190219BHEP

Ipc: A61K 31/519 20060101ALI20190219BHEP

Ipc: A61K 31/337 20060101ALI20190219BHEP

Ipc: A61K 31/704 20060101ALI20190219BHEP

Ipc: A61K 31/4745 20060101ALI20190219BHEP

Ipc: A61K 45/06 20060101AFI20190219BHEP

Ipc: A61K 47/65 20170101ALI20190219BHEP

Ipc: G01N 33/50 20060101ALI20190219BHEP

Ipc: A61K 31/537 20060101ALI20190219BHEP

Ipc: A61K 31/475 20060101ALI20190219BHEP

INTG Intention to grant announced

Effective date: 20190327

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1177626

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190915

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602013060467

Country of ref document: DE

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: DENNEMEYER AG, CH

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20191122

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191211

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191211

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20191212

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1177626

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190911

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200113

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2759999

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20200512

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200224

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602013060467

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG2D Information on lapse in contracting state deleted

Ref country code: IS

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200112

26N No opposition filed

Effective date: 20200615

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20191227

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20131227

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190911

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230523

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20230530

Year of fee payment: 10

Ref country code: IT

Payment date: 20230530

Year of fee payment: 10

Ref country code: IE

Payment date: 20230530

Year of fee payment: 10

Ref country code: FR

Payment date: 20230530

Year of fee payment: 10

Ref country code: CH

Payment date: 20230629

Year of fee payment: 10

Ref country code: ES

Payment date: 20230529

Year of fee payment: 10

Ref country code: DK

Payment date: 20230531

Year of fee payment: 10

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20230601

Year of fee payment: 10

Ref country code: PL

Payment date: 20230602

Year of fee payment: 10

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20230530

Year of fee payment: 10

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20231229

Year of fee payment: 11

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20231229

Year of fee payment: 11

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20231231

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20240101

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20231231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240101

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240101

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231227

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231231